Method of stabilizing human eye tissue

ABSTRACT

Disclosed are compounds and related methods useful for cross-linking collagen and stabilizing collagenous tissues using formaldehyde-donating compounds or nitrogen oxide-containing compounds such as β-Nitro Alcohols. Also disclosed are compounds and related methods for modulating the rate or degree of collagen cross-linking using nitrogen oxide-containing compounds such as β-Nitro Alcohols. The formaldehyde-donating, nitrogen oxide-containing and/or β-Nitro Alcohol compounds disclosed are capable of stabilizing collagenous tissues such as the corneal and scleral tissues and are useful in the treatment or prevention of diseases such as alterations in corneal curvature, keratoconus, keratectasia, progressive myopia and glaucoma.

RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. patent applicationSer. No. 12/517,382, filed on Nov. 10, 2009, which is a national stagefiling under 35 U.S.C. 371 of International Application No.PCT/US2007/025126, filed Dec. 6, 2007, which claims the benefit under 35U.S.C. 119(e) of Provisional Application No. 60/873,353 filed on Dec. 6,2006, and Provisional Application No. 60/936,635 filed Jun. 20, 2007.The entire teachings of the above applications are incorporated hereinby reference.

GOVERNMENT SUPPORT

This invention was made with government support under United StatesGovernment Grant Nos. K08 AG00863, R01 HL075639 and R01 EY020495 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

Collagen is a fundamental protein found in connective tissue in animals,and it is present in the cornea and sclera of the eye. Several eyedisorders are related to defects in collagen structure and includekeratoconus, keratectasia, progressive myopia, and possibly glaucoma.

Keratoconus is a debilitating, progressive eye disorder, which isbelieved to occur due to progressive slippage of collagen lamellae inthe cornea, usually bilateral, beginning between ages 10 and 20. Thecornea develops a conical shape, causing significant changes in therefractive power of the eye. While corrective lenses may help vision,corneal transplant surgery may be necessary if eyeglasses or contactlenses are inadequate. THE MERCK MANUAL OF DIAGNOSIS AND THERAPY 722(Mark H. Beers and Robert Berkow eds., 17th ed. 1999).

Keratoconus is estimated to affect 1 person in about 435 to 2000 peoplein the general population. In its classical form, keratoconus commencesat puberty and progresses into the third to fourth decade of lifeRabinowitz, Y. S. “Keratoconus,” Surv. Opthal. 1998; 43(4):297-319.Thus, its overall impact is magnified by virtue of the youngerpopulation that it afflicts. Clinically, the disease is marked byprogressive thinning of the corneal stroma with resultant bulging anddistortion of the thinned, weakened areas. This thinning and distortionis documented by optical and ultrasonic methods. The bulging, distortedcornea creates an optically imperfect surface to the eye that producesan increasingly irregular astigmatism and myopia. Contact lenses areused to correct these optical imperfections when spectacle lenses are nolonger able to compensate for the induced optical distortion. Whencontact lens correction fails, only a corneal transplant will allowrestoration of visual function. The need for corneal transplantationarises when the disease has progressed and central corneal scarformation occurs, or the distortion is so great that contact lenses canno longer be worn.

Although the underlying etiology of keratoconus remains unclear, thereare two main mechanistic theories currently entertained. The first isrelated to destabilization of collagen lamellae through increaseddegradation via imbalances in endogenous proteases and/or theirinhibitors. In this regard, the scientific evidence has been somewhatequivocal with some studies showing increased matrix-metalloproteinaseactivity and others reporting no change (reviewed by Collier, S. A., “Isthe corneal degradation in keratoconus caused bymatrix-metalloproteinases?” Clin. Exp. Ophthalmol. 2001; 29:340-344). Analternative theory regards collagen fibril slippage with no overalltissue loss. Meek, K. M., et al. have shown, using synchrotron X-rayscattering, that stromal lamellar organization is altered with anassociated uneven distribution of collagen fibrillar mass. These changesare consistent with inter- and/or intra-lamellar slippage within thestromal layers of the keratoconic cornea, leading to central thinning.Meek, K. M., et al., “Changes in collagen orientation and distributionin keratoconus corneas,” IOVS 2005; 46(6):1948-1956. The defect thatwould allow such slippage could be related to changes in the collagen toproteoglycan interactions and/or qualitative changes in the fibrillarcollagens. Regarding this second point, very little is known about thequalitative biochemical collagen changes that occur in keratoconus.However, alterations in difunctional collagen cross-linking werereported decades ago. Cannon, J. and Foster, C. S., “Collagencrosslinking in keratoconus,” IOVS 1978; 17(1):63-65; Oxlund, H. andSimonsen, A. H., “Biochemical studies of normal and keratoconuscorneas,” 1985; 63:666-669; Critchfield, J. W., et al., “Keratoconus: I.biochemical studies,” Exp. Eye Res. 1988; 46:953-963. Regardless of theexact mechanism responsible for progressive corneal thinning, thepathologic changes that take place are accompanied by a loss ofbiomechanical strength. In this regard it has been shown thatkeratoconic corneas show a decreased stress for a given strain ascompared to controls (i.e., decreased tissue stiffness) [Andreassen, T.T., et al., “Biomechanical properties of keratoconus and normalcorneas,” Exp. Eye Res. 1980; 31:435-441.] Andreassen, T. T., et al.also found that keratoconus collagen displayed a decreased resistance toenzymatic digestion with pepsin, a finding which is consistent withalterations in collagen cross-linking.

Current treatments for keratoconus either mask the surface irregularitywith a variety of contact lenses, or attempt to improve the surfacecontour with intracorneal ring segments, lamellar keratoplasty, orexcimer laser surgery. Binder, P. S., et al., “Keratoconus and cornealectasia after LASIK,” J. Refract. Surg. 2005; 21:749-752. However, thedisease is progressive and none of these options obviates the need foreventual corneal transplantation.

Glaucoma is a group of disorders characterized by progressive damage tothe eye at least partly due to increased intraocular pressure, theaqueous pressure in the eye. Increased intraocular pressure results froman inadequate aqueous outflow from the eye due to an obstruction in thetrabecular meshwork from which the eye drains. Collagen is necessary tomaintain the structural integrity of the trabecular meshwork. Rehnberg,M., et al., “Collagen distribution in the lamina cribosa and thetrabecular meshwork of the human eye.” Brit. J. Ophthalmol. 71:886-92(1987). Open-angle glaucoma can be treated with medical, laser, orsurgical therapy to prevent damage to the optic nerve and visual fieldby stabilizing the intraocular pressure. THE MERCK MANUAL OF DIAGNOSISAND THERAPY 733-36 (Mark H. Beers and Robert Berkow eds., 17th ed.1999).

In myopia, or nearsightedness, the image of a distant object is focusedin front of the retina because the axis of the eyeball is too long orthe refractory power of the eye is too strong. Rays of light fall infront of the retina because the cornea is too steep or the axial lengthof the eye is too long. Without glasses, distant images are blurry, butnear objects can be seen clearly. While glasses or contact lensescorrect vision, refractive surgery decreases a patient's dependence onglasses or contact lenses. Progressive myopia is a condition associatedwith high refractive error and subnormal visual acuity after correction.This form of myopia gets progressively worse over time. THE MERCK MANUALOF DIAGNOSIS AND THERAPY 741-43 (Mark H. Beers and Robert Berkow eds.,17th ed. 1999). The development of severe myopia is associated withscleral thinning and changes in the diameter of scleral collagen fibrilsin humans. McBrien, N. A., et al., “Structural and UltrastructuralChanges to the Sclera in a Mammalian Model of High Myopia.”Investigative Ophthelmol. & Visual Sci. 42:2179-87 (2001).

Refractive surgery alters the curvature of the cornea to allow lightrays to come to focus closer to the retina, thus improving uncorrectedvision. In myopia, the central corneal curvature is flattened. However,ideal candidates for refractive surgery are people with healthy eyes whoare not satisfied wearing glasses or contact lenses for their daily orrecreational activities. Candidates for refractive surgery should nothave a history of collagen vascular disease because of potentialproblems with wound healing. As keratoconus is a progressive thinning ofthe cornea, thinning the cornea further with refractive surgery maycontribute to the advancement of the disease. Huang, X., et al.,“Research of corneal ectasia following laser in-situ keratomileusis inrabbits.” Yan Ke Xue Bao, 18(2):119-22 (2002), The side effects ofrefractive surgery include temporary foreign-body sensation, glare, andhalos. Potential complications include over- and under-correction,infection, irregular astigmatism, and, in excimer laser procedures, hazeformation. Permanent changes in the central cornea caused by infection,irregular astigmatism, or haze formation could result in a loss of bestcorrected acuity.

Keratectasia is the protrusion of a thinned, scarred cornea. In laser insitu keratomileusis (LASIK), if the laser removes too much tissue, orthe flap is made too deep, the cornea can become weak and distorted,leading to keratectasia. LASIK is contraindicated for patients with thincorneas, or those with keratectasia as a result of a prior LASIKprocedure. Rigid gas permeable contact lenses are the recommendedtreatment for correcting vision in these patients. Kim, H., et al.,“Keratectasia after Laser in situ Keratomileusis.” Int'l, J. Ophthalmol.220:58-64 (2006).

A major breakthrough in the treatment of keratoconus and relatedkeratectasias has been realized. Recent work by the German group ofWollensak, Spoerl, and Seiler has shown that cross-linking cornealcollagen through application of riboflavin and ultraviolet light (UVR)can limit progressive vision loss in keratoconus patients. This modalityrepresents a method through which stabilization of the corneal collagenlamellae and has been shown to prevent the progressive thinning of thecornea and loss of vision observed in keratoconus patients. Thistreatment involves the serial applications of riboflavin (0.1%) onto ade-epithelialized human cornea followed by exposure of the riboflavinsaturated tissue to ultraviolet radiation in a UVA-370 nanometerwavelength region, at 3 mW/cm² radiant energy. The patient is treatedwith antibiotic drops to prevent infection and oral pain medicine afterthe procedure. Literature accruing over the past 9 years has describedthe utility of photochemical cross-linking using UVA irradiation(2λmax=370 nm) with riboflavin as a photosensitizer (UVR). The work ofthe German group of Wollensak, G., Spoerl, E., and Seiler T., has shownthat this method of cross-linking the collagen within the corneal stromahas proven effective in limiting the progression of corneal thinning,distortion, and resulting optical degradation of the eye. Wollensak, G.,et al., “Riboflavin/ultraviolet-A-induced collagen crosslinking for thetreatment of keratoconus.” Am. J. Ophthalmol. 2003; 135:620-27. Despitethese successes, the UVR therapy poses attendant risks, particularlyrelated to ultraviolet irradiation. As such, this therapy has yet togain FDA approval in the US.

Because riboflavin tissue penetration is limited by the cornealepithelium, it is necessary to remove the corneal epithelium by scrapingprior to riboflavin application. Removal of the corneal epitheliumexposes the cornea to a risk of infection and causes significant pain.In addition, keratocyte (Wollensak, G., et al., “T. keratocytecytotoxicity of riboflavin/UVA treatment in vitro.” Eye, 18:718-22(2004); Wollensak, G., et al., “Keratocyte apoptosis after cornealcollagen cross-linking using riboflavin/UVA treatment.” Cornea,23(1):43-49 (2004)) and corneal endothelial cell toxicity (Wollensak,G., et al., “Corneal endothelial cytotoxicity of riboflavin/UVAtreatment in vitro.” Ophthalmic Res., 35:324-28 (2003)) can occur withapplication of riboflavin/UVA to the cornea. In a similar manner,application of this therapy to the posterior sclera has been reported todamage cells in the photoreceptor, outer nuclear, and retinal pigmentepithelial layers (Wollensak, G., et al., “Cross-linking of scleralcollagen in the rabbit using riboflavin and UVA,” Acta OphthalmologicaScandinavica, 83:477-82 (2005).

Currently, clinical trials are ongoing in Europe (Caporossi, A., et al.,“Parasurgical therapy for keratoconus by riboflavin-ultraviolet type Arays induced cross-linking of corneal collagen: Preliminary refractiveresults in an Italian study,” J. Cataract Refract. Surg. 2006;32:837-845; Wollensak, G., “Crosslinking treatment of progressivekeratoconus: new hope,” Cur. Opin. Ophthal. 2006; 17:356-360) withsignificant interest generated for initiating clinical trials in theUnited States. The early reports from this therapy are encouraging.After 5 years in the Dresden study, individuals who have undergone thistreatment protocol have yet to show progression of their keratoconus.With these encouraging results, corneal cross-linking therapy is beingextended to include patients with related disorders such as the ectasiathat occurs following LASIK (Laser-Assisted In situ Keratomileusis) andPRK (Photorefractive Keratectomy) excimer refractive surgery (Binder, P.S., et al., 2005). These are devastating complications ofkeratorefractive surgery in today's clinical practice. Anecdotal reportshave also emerged reporting the use of collagen cross-linking as aneffective means to control difficult-to-treat corneal fungal infectionsand corneal melts.

Despite these successes, the UVR therapy poses attendant risks,particularly related to ultraviolet irradiation. As such, this therapyhas encountered difficulty gaining FDA approval and is currentlyunavailable in the United States. Because free oxygen radical formationoccurs with riboflavin photolysis (Baier, J., et al., “Singlet oxygengeneration by UVA light exposure of endogenous photosensitizers,”Biophys. J. 2006; 91:1452-1459), this cross-linking method has anegative impact on cell viability. Indeed, keratocyte (Wollensak, G., etal., 2004) and corneal endothelial cell toxicity (Wollensak, G., et al.,2003) does occur with application of this therapy to the cornea. As aresult of such toxicity, it has been recommended that patients withparticularly thin central corneas (<40 μm) not undergo this therapysince the depth of UVA penetration exposes the endothelial cells (whichare vital to maintaining corneal clarity through water regulation) totoxic photochemical damage. Furthermore, the long-term risks of thisphotochemical exposure are not known. Secondly, deep tissue penetrationby the riboflavin requires removal of the corneal epithelium, aprocedure that increases morbidity and complications. This requiresanalgesics and antibiotics following the UVR cross-linking procedure.

A need in the art exists to develop a topical self-administered compoundin order to produce a comparable degree of collagen cross-linking to theUVR therapy. Also needed are methods and compositions for modulating(e.g., increasing or decreasing) the rate or degree at which suchcompounds effectuate collagen cross-linking.

SUMMARY OF THE INVENTION

This invention generally provides methods of cross-linking collagen in acollagenous tissue of a subject in need thereof comprising contactingthe collagenous tissue with an amount of a formaldehyde-donatingcompound (e.g., a nitrogen oxide-containing compound, such as one ormore nitro alcohols, β-nitro alcohols or βNAs) effective to cross-linkthe collagen in the collagenous tissue.

The nitrogen oxide-containing compounds disclosed herein (e.g., βNAssuch as 2-nitroethanol, 2-nitro-1-propanol, 3-nitro-2-pentanol and/or2-nitro-1-pentanol) may induce or otherwise facilitate the cross-linkingof collagen (e.g., collagen in a collagenous tissue), thereby makingthem suitable candidates for in vivo (e.g., ophthalmic) administration.Additionally, formaldehyde-donating compounds, such as βNAs generallydemonstrate low toxicity, thereby making them amenable to clinical use(e.g., in vivo ophthalmic administration for the treatment ofkeratoconus or keratectasia.)

Disclosed herein are the underlying mechanisms by whichformaldehyde-donating compounds or nitrogen oxide-containing compounds(e.g., nitro alcohols, such as βNAs) induce, facilitate or otherwisecause the cross-linking of collagen (e.g., collagen found in collagenoustissues). βNAs (e.g., one or more ofhydroxymethyl-2-nitro-1,3-propanediol, 2-methyl-2-nitro-1,3-propanedioland 2-bromo-2-nitro-1,3-propanediol) may be prepared by way of thereversible nitro-aldol condensation reaction (the Henry Reaction). Asillustrated in FIG. 26, the Henry Reaction is reversible; therefore βNAssubject to the reverse Henry Reaction are capable of being converted to,or otherwise producing an aldehyde species (e.g., formaldehyde) and anitroalkane. The present inventors have demonstrated that the aldehydespecies (e.g., formaldehyde) directly participate in the cross-linkingof collagen.

Accordingly, also disclosed herein are methods and compositions formodulating (e.g., accelerating or decelerating) the rate at whichformaldehyde-donating compounds (e.g., nitro alcohols, such as βNAcompounds) cross-link collagen. In the presence of certain conditions(e.g., alkaline conditions) or catalysts (e.g., salmon sperm DNA), thereverse Henry Reaction can be promoted or otherwise stimulated therebypromoting the formation of an aldehyde species (e.g., formaldehyde) fromthe βNAs and consequently promoting the cross-linking of collagen. Nitroalcohol, formaldehyde-donating compounds, such as βNAs (e.g.,2-bromo-2-nitro-1,3-propanediol) may therefore serve as a formaldehydedonor (e.g., in an alkaline environment) capable of cross-linkingcollagenous tissues.

The present inventions also disclose compositions and methods forcatalyzing the cross-linking of collagenous tissues. For example, nitroalcohols, such as βNA compounds may be administered to a subject (e.g.,a mammal) in the presences of one or more catalysts (e.g., bases, DNAand/or enzymes) as a means of promoting, accelerating or catalyzing thecross-linking of collagen (e.g., collagen in collagenous tissues such asthe skin.) Contemplated catalysts capable of driving the reverse HenryReaction, thereby promoting the formation of an aldehyde species fromthe nitro alcohol (e.g., βNA), include exposure to basic or alkalineconditions (e.g., exposure to NaHCO₃), single or double stranded DNA(e.g., salmon testes DNA) and/or enzymes (e.g., hydroxynitrile lyases,transglutaminases and hydrolases.)

In certain embodiments, the present inventions relate to methods ofaccelerating the rate at which one or more formaldehyde-donatingcompounds (e.g., nitro alcohols, such as βNAs) cross-link collagen(e.g., in a collagenous tissue.) Such methods generally comprise a stepof contacting or otherwise exposing one or more formaldehyde-donatingcompounds (e.g., nitro alcohols, such as βNAs) with or to one or morecatalysts. In certain embodiments the one or more catalysts are capableof accelerating the rate at which an aldehyde species (e.g.,formaldehyde) is formed or otherwise produced from the one or moreformaldehyde-donating compounds (e.g., βNAs), thereby accelerating therate at which the collagen is cross-linked in the collagenous tissue(e.g., cornea, sclera, or skin.) In some embodiments, such catalystspromote or otherwise catalyze the reverse Henry Reaction.

The methods, compositions and compounds disclosed herein may compriseone or more formaldehyde-donating compounds (e.g., βNAs.) In certainembodiments, the formaldehyde-donating compound is a nitro alcohol. Forexample, in certain embodiments the βNA may comprise one or more of2-nitro-1-propanol, 2-nitro-1-pentanol, 3-nitro-2-pentanol,2-hydroxymethyl-2-nitro-1,3-propanediol,2-methyl-2-nitro-1,3-propanediol, 2-bromo-2-nitro-1,3-propanediol orcombinations thereof.

In certain instances, the step of contacting one or moreformaldehyde-donating compounds (e.g., βNAs) with one or more catalystsis performed in the presence of collagenous tissue. For example, one ormore βNAs and one or more catalysts may be co-administered to a subject(e.g., a human) either simultaneously or in succession. Alternatively,in other instances the step of contacting one or moreformaldehyde-donating compounds (e.g., nitro alcohols, such as βNAs)with one or more catalysts is performed in the absence of thecollagenous tissue. For example, one or more βNAs and one or morecatalysts may be admixed prior to contacting a collagenous tissue.

In certain embodiments, the selected catalyst may comprise bases (e.g.,one or more bases selected from the group consisting of aqueoussolutions of NaHCO₃, KOH, NaOH, Ca(OH)₂, Na₂CO₃, KF, Bu₃P, MeONa/MeOH,Et₃N and combinations thereof.) In some embodiments the selectedcatalyst may comprise DNA (e.g., salmon sperm DNA.) Similarly, in otherembodiments the selected catalysts comprise enzymes.

In some embodiments, the one or more catalysts accelerate the rate atwhich collagen is cross-linked. For example, relative to the rate atwhich one or more formaldehyde-donating compounds (e.g., βNAs)cross-link collagen in the absence of a catalyst, the catalystsdisclosed herein may increase the rate at which one or more βNAscross-link collagen by at least about two-, three-, four-, five-, six-,seven-, eight-, nine-, ten-, twelve-, fifteen-, eighteen-, twenty-,twenty five-, thirty-, fifty, one hundred-fold, or more.

Conversely, in other embodiments, the catalysts disclosed herein (e.g.,hydroxynitrile lyases, transglutaminases and hydrolases) may deceleratethe rate at which collagen is cross-linked. For example, such catalystsmay drive or otherwise promote the Henry Reaction, thereby promoting theformation of βNs and suppressing the formation of aldehyde species(e.g., formaldehyde.) Therefore, also disclosed herein are methods ofdecelerating the rate at which one or more formaldehyde-donatingcompounds, such as βNAs, cross-link collagen in a collagenous tissue.Such methods generally comprising a step of contacting one or moreformaldehyde-donating compounds (e.g., βNAs) with one or more enzymes(e.g., hydroxynitrile lyases, transglutaminases and hydrolases) capableof decelerating the rate at which formaldehyde is formed from one ormore βNAs, thereby decelerating the rate at which collagen iscross-linked (e.g., in collagenous tissue.) Relative to the rate atwhich formaldehyde-donating compounds (e.g., βNAs) cross-link collagenin the absence of an enzyme, in certain embodiments such enzymesdecelerate the rate at which collagen is cross-linked (e.g., incollagenous tissue) by at least about two-, three-, four-, five-, six-,seven-, eight, nine-, ten-, twelve-, fifteen-, eighteen-, twenty-,twenty five-, thirty-, fifty, one hundred-fold, or more.

The present invention also relates to methods of treating a conditionassociated with a loss of structural integrity of collagenous tissues(e.g., cornea, sclera, blood vessels, cartilage, tendon, bone and/orskin.) For example, such conditions may include, but not be limited to,keratoconus, keratectasia and myopia. Such methods generally comprise astep of contacting the collagenous tissue with a composition comprisingone or more formaldehyde-donating compounds (e.g., nitro alcohols orβNAs) and one or more catalysts, wherein the composition cross-links thecollagen in the collagenous tissue thereby improving the loss ofstructural integrity of such collagenous tissue.

In certain embodiments, the selected catalysts are capable of promotingthe reverse Henry Reaction and thereby accelerating collagencross-linking in the presence of one or more formaldehyde-donatingcompounds (e.g., βNAs). For example, exposure of βNAs to basic oralkaline conditions (e.g., a pH equal or greater than about 7.0) may beused as a means of promoting the reverse Henry Reaction. Accordingly, incertain embodiments the catalyst comprises a base (e.g., a base selectedfrom the group consisting of aqueous solutions of NaHCO₃, KOH, NaOH,Ca(OH)₂, Na₂CO₃, KF, Bu₃P, MeONa/MeOH, Et₃N and combinations thereof) oran enzyme. Similarly, exposure of βNAs to DNA (e.g., salmon sperm DNA)may promote the reverse Henry Reaction. In some embodiments, theselected catalysts accelerate the cross-linking of collagen (e.g., incollagenous tissue) by the formaldehyde-donating compounds (e.g., βNA)by at least about two-, three-, four-, five-, six-, seven-, eight-,nine-, ten-, twelve-, fifteen-, eighteen-, twenty-, twenty five-,thirty-, fifty, one hundred-fold, or more relative to the rate at whichthe βNAs cross-link collagen in the absence of such catalysts.

In certain embodiments, the selected catalysts are capable of promotingthe forward Henry Reaction and thereby decelerating the rate at whichcollagen cross-links in the presence of βNAs. For example, exposure ofβNAs to certain enzymes (e.g., hydroxynitrile lyase) may promote theforward Henry Reaction. Accordingly, in certain embodiments the catalystcomprises one or more enzymes (e.g., hydroxynitrile lyases,transglutaminases and hydrolases). In some embodiments, the selectedcatalysts decelerate the cross-linking of collagen (e.g., in collagenoustissues) by the βNA by at least about two-, three-, four-, five-, six-,seven-, eight-, nine-, ten-, twelve-, fifteen-, eighteen-, twenty-,twenty five-, thirty-, fifty, one hundred-fold, or more relative to therate at which the βNAs cross-link collagen in the absence of suchcatalyst.

The present inventions also relate to methods of cross-linking collagenin a collagenous tissue (e.g., the skin of a mammal). Such methodsgenerally comprise a step of contacting the collagenous tissue (e.g.,the cornea, sclera or skin of a human subject) with a composition (e.g.,a composition formulated for ophthalmic or topical administration)comprising one or more formaldehyde-donating compounds (e.g., nitroalcohols or β-nitro alcohols) and one or more catalysts, wherein thecomposition effectuates the cross-linking of the collagen in thecollagenous tissue.

In some embodiments disclosed herein, the catalyst comprises a base(e.g., one or more bases selected from the group consisting of NaHCO₃,KOH, NaOH, Ca(OH)₂, Na₂CO₃, KF, Bu₃P, MeONa/MeOH, Et₃N and combinationsthereof.) In some embodiments the catalyst comprises DNA (e.g., salmonsperm DNA.) In yet other embodiments the catalyst comprises an enzyme.

In certain embodiments, the catalysts (e.g., bases, DNA and/or enzymes)promote the reverse Henry Reaction and thereby accelerate thecross-linking of collagen by the βNA by at least about two-, three-,four-, five-, six-, seven-, eight-, nine-, ten-, twelve-, fifteen-,eighteen-, twenty-, twenty five-, thirty-, fifty, one hundred-fold, ormore relative to the rate at which the formaldehyde-donating compounds(e.g., a nitro alcohol or βNA) cross-links collagen in the absence ofsuch catalysts. In certain alternative embodiments, the selectedcatalysts (e.g., the enzyme hydroxynitrile lyase) promote the forwardHenry Reaction, thereby decelerating the rate at which the βNAscross-link collagen (e.g., in a collagenous tissue.)

In certain embodiments, the formaldehyde-donating compounds for use inthe methods or compositions disclosed herein are βNAs selected from thegroup consisting of 2-hydroxymethyl-2-nitro-1,3-propanediol,2-methyl-2-nitro-1,3-propanediol, 2-bromo-2-nitro-1,3-propanediol,2-nitroethanol, 2-nitro-1-propanol, 3-nitro-2-pentanol,2-nitro-1-pentanol and combinations thereof.

In certain embodiments, the methods and compositions disclosed hereinare suitable for administration to, or for the treatment of a subject(e.g., a human) afflicted with a condition selected from the groupconsisting of keratoconus, keratectasia and myopia.

The compounds and compositions disclosed herein may be formulated foradministration to a subject (e.g., a mammal) using any suitable routesof administration (e.g., orally, parenterally, intramuscularly,subcutaneously, ophthalmically, topically, nasally or transdermally.) Incertain embodiments, such compounds and compositions are administeredophthalmically. Similarly, the compositions disclosed herein (e.g.,compositions intended for topical or ophthalmic administration) may beprepared or reconstituted in any suitable dosage form (e.g., a solution,a suspension, an ointment, a gel or a cream.) In certain embodiments,the compounds and compositions disclosed herein may be formulated forextended- or delayed-release of one or more of the constituent compounds(e.g., formaldehyde-donating compounds such as βNAs.) For example,following the administration of such compositions to a subject one ormore of the βNAs and/or catalysts that comprise such composition may bereleased over an extended period of time (e.g., released over the courseof at least about 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours,12 hours, 16 hours, 18 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 1week or more.)

In certain embodiments, such compositions have an alkaline pH (e.g., apH between about 7 and 10.) In other embodiments, such compositions areformulated such that they approximate physiologic pH (e.g., a pH ofabout 7.4.) In yet other embodiments, such compositions are formulatedsuch that they have an acidic pH (e.g., a pH less than about 7) orweakly acidic pH (e.g., a pH between about 5 and 7.)

Also disclosed herein are methods of inhibiting the loss of structuralintegrity of a collagenous tissue during transplantation relatedtransport, such methods generally comprising a step of contacting thecollagenous tissue with an amount of a nitrogen oxide containingcompound (e.g., one or more βNAs) effective to inhibit loss ofstructural integrity of the collagenous tissue.

This invention further provides a composition for ophthalmicadministration comprising a formaldehyde-donating compound (e.g., anitrogen oxide containing compound such as a βNA), sodium chloride,potassium chloride, calcium chloride dihydrate, magnesium chloridehexahydrate, sodium acetate trihydrate, sodium citrate dehydrate, andwater.

Also provided herein, are methods of altering the refractive power of asubject's cornea. Such methods generally comprise a step of contactingthe cornea with a formaldehyde-donating compound or a nitrogenoxide-containing compound (e.g., a nitro alcohol such as a βNA) so as toeffect cross-linking of collagen in the cornea and thereby alter therefractive power of the cornea.

The above-discussed and many other features and attendant advantages ofthe present invention will become better understood by reference to thefollowing detailed description of the invention when taken inconjunction with the accompanying examples. The various embodimentsdescribed herein are complimentary and can be combined or used togetherin a manner understood by the skilled person in view of the teachingscontained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1—Protein concentration of collagen from nitrite treated cells insupernatant (μg/μl).

FIG. 2—(A) SDS-PAGE (5% separating gel) of post-treated type I collagenfollowing a 7-day incubation (pH 7.4) with 0, 10, 100 mM NaNO2, and 100mM NaCl, (B) Densitrometric analysis of FIG. 2(A) gel, (C) Biomechanicaltesting on fibroblast populated collagen type I gels.

FIG. 3—Fibroblast populated fibrillar collagen gels for biomechanicaltesting.

FIG. 4—Diagram and average deformation data showing the phases of theexperiment for a uniaxially constrained gel.

FIG. 5—Impact of sodium nitrite on vertical deformation of uniaxiallyconstrained gels during each phase of the experiment. (A) Pretreatmentat pH 4.0 for 24 hours with 100 mM sodium nitrite and post-treatment atpH 7.4 for seven days with 100 mM sodium nitrite both reduced verticalremodeling; (B) Pretreatment at pH 4.0 for 24 hours with 100 mM sodiumnitrite and post-treatment at pH 7.4 for seven days with 100 mM sodiumnitrite both reduced vertical deformation associated withpreconditioning; and (C) Pretreatment at pH 4.0 for 24 hours with 100 mMsodium nitrite and post-treatment at pH 7.4 for seven days with 100 mMsodium nitrite both reduced vertical deformation at 1 g equibiaxialload.

FIG. 6—Passage of nitrite into the anterior chamber at 24 hours, pH 7.4,and 4° C.

FIG. 7—Porcine cornea treated with 200 mM NaNO₂, pH 5.0, 37° C., for 30hours and untreated porcine cornea.

FIG. 8—Porcine sclera treated with 200 mM NaNO₂, pH 5.0, 37° C., for 30hours and untreated porcine sclera.

FIG. 9—Percent shrinkage of porcine cornea treated with 200 mM NaNO₂, pH5.0, 37° C., for 30 hours and 80 hours, and untreated porcine cornea,with 2° C. temperature increase every five minutes.

FIG. 10—Custom box for shrinkage temperature analysis, a general measureof collagen cross-linking.

FIG. 11—Example of shrinkage temperature changes in cross-linked porcinesclera.

FIG. 12—Planar biaxial material testing system with sample loaded incenter.

FIG. 13—Stiffening effects in corneal tissue induced by reaction withβ-nitro alcohols. Fresh porcine corneal strips and corneoscleralcomplexes obtained within 6 his of sacrifice and incubated at 37° C. inbuffered solutions (pH 7.4) containing 20% Dextran (T500) and either 100mM NaNO₂ or 100 mM of one each of the specified β-nitro alcohols: (A)2-nitroethanol, (B) 2-nitro-1-propanol, (C) 3-nitro-2-pentanol, (D)NaNO₂ or (E) control.

FIG. 14—Corneal transparency is preserved following cross-linking withβ-nitro alcohols. Corneal tissue induced by reaction with 100 mM of: (A)2-nitroethanol, (B) 2-nitro-1-propanol, (C) β-nitro-2-pentanol, wherein,the sample mounted over the letter “C” is control and the sample mountedover the letter “N” is the specified β-nitro alcohol.

FIG. 15—Alterations in light transmission induced by cornealcross-linking with β-nitro alcohols. Following a 96 hour incubationperiod at 10 mM, corneo-scleral complexes were mounted for measurementof absorbance spectra. Decreased transmission was greatest for2-nitroethanol, followed by 2-nitro-1-propanol, and 3-nitro-2-pentanol.Integration of the 400-500 nm blue light region revealed decreases of3.6%, 1.5%, and 1.0% respectively. Sample of the analysis andAbsorbance/transmission curves are shown.

FIG. 16—Thermal shrinkage is increased in porcine cornea throughcross-linking by β-nitro alcohols. Specified β-nitro alcohols wereserially applied over 6 days using various concentrations (1-100 mM) of:(A) 2-nitroethanol, (B) 2-nitro-1-propanol or (C) 3-nitro-2-pentanol.

FIG. 17—Concentration dependent effects on shrinkage temperature curvesby four different β-nitro alcohols. Following 96 hrs of incubation at37° C. using various concentrations of the specified β-nitro alcohols(1-100 mM), thermal shrinkage temperature was determined. (A)2-nitroethanol, (B) 2-nitro-1-propanol, (C) 3-nitro-2-pentanol, and (D)2-nitrophenol.

FIG. 18—Concentration dependent shift in T_(s) of human sclera using2-nitroethanol. Following a 96 hrs incubation using variousconcentrations of 2-nitroethanol (1-100 mM), T_(s) was determined. T₅₀was shifted 0.3, 2.2, and 7.5° C. using 1, 10, and 100 mM2-nitroethanol, respectively. The concentration dependent effectobserved was similar to that observed using porcine sclera as in (B),which is the same graph shown as FIG. 18A (included for comparisonpurposes). (A) Human and (B) Porcine.

FIG. 19—Time dependent shift in T_(s) using 2-nitroethanol at 100 mM and1 mM. (A) 100 mM 2-nitroethanol: Time of incubation was varied from 24to 96 hrs. A time dependent effect in thermal shrinkage temperatureshift was noted for 2-nitroethanol at 100 mM concentration over thecourse of 96 hrs with T₅₀ shifts of 1.4, 2.4, 5.3, and 7.7° C. for 24,48, 72, and 96 hrs of incubation, respectively. (B) 1 mM 2-nitroethanol:Time dependent shift in T_(s) using 2-2-nitroethanol at 1 mM. Conditionswere as in (A) except that the time of incubation was varied from 0 to14 days using a concentration of 1 mM. In addition, the incubationsolution was “exchanged daily” using a 1 mM solution of 2-nitroethanol.A time dependent shift in T_(s) was noted for 2-nitroethanol at 1 mMconcentration over the course of 14 days. T₅₀ was shifted 1.3, 3.2, and5.6° C. for 6, 10 and 14 days of incubation, respectively. The shift inT_(s) was commensurate to the shift observed using higher concentrationsof 2-nitroethanol (i.e. 10 and 100 mM) for shorter durations (i.e. 24-96hrs) [see FIG. 18A].

FIG. 20—Degrees of crosslinking through modulation of reagentconcentration and time of exposure.

1 mM 2-nitroethanol concentration, the incubation solution was changeddaily over the course of 10 days and compared to Ts changes producedthrough incubating with 10 mM 2-nitroethanol over 4 days and 100 mM2-nitroethanol over 3 days without changing the solution.

FIG. 21—Shrinkage temperature curve changes using nitroethane.

FIG. 22—Shrinkage temperature curve changes using isopentyl nitrite onporcine sclera.

FIG. 23—Shrinkage temperature curve changes using (A) DPTA and (B) DETAon porcine sclera.

FIG. 24—Initial toxicity studies using ARPE-19 (24 hour exposure). (A)Control, (B) 1 mM 2-nitroethanol, (C) 100 mM NaNO₂, and (D) 10 mM2-nitroethanol.

FIG. 25—Toxicity levels vary between β-nitro alcohols Toxicity studiesin primary cultures of bovine corneal endothelial cells. (A) 1 mM2-nitroethanol, (B) 1 mM 2-nitro-1-propanol, (C) 1 mM 3-nitro-2-pentanoland (D) Control.

FIG. 26—Illustrates the reversible nitro-aldol condensation reaction(the Henry Reaction) whereby a nitroalkane reacts with an aldehyde toform a β-nitro alcohol (βNA) product. As shown in FIG. 26, in thepresence of base (B) the βNA undergoes de-protonation at the hydroxylgroup, followed by the subsequent decomposition of the βNA to yieldformaldehyde by a base-catalyzed reverse Henry reaction.

FIG. 27—Depicts the mechanism by which β-nitro alcohols (βNAs) crosslinka poly(allyamine) (PAA) substrate, which was used as a surrogate modelfor determining the ability of βNAs to cross-link collagen in vivo. Asillustrated in FIG. 27, an amino group of the PAA side chain with thelone electron pair attacks the electrophilic carbonyl carbon offormaldehyde from the βNA to form a Schiff base, which can then reactwith another PAA and formaldehyde to crosslink the PAA.

FIG. 28—Three kinds of gels during the swelling process are depicted,including hydrogel before being washed and dried (left), and after beingwashed and dried (middle) and the swollen hydrogel (right).

FIG. 29—Depicts an ATR-FTIR of a poly(allyamine) (PAA) substratecross-linked with paraformaldehyde (PFA) at pH 7.4 and 37° C. bothbefore (----) and after (

) being washed with ethanol and water and dried.

FIG. 30—Depicts an ATR-FTIR of a poly(allyamine) (PAA) substratecross-linked with 2-hydroxymethyl-2-nitro-1,3-propanediol (nitro-triol)at pH 7.4 and 37° C. both before (----) and after (

) being washed with ethanol and water and dried.

FIG. 31—Illustrates the ¹H-NMR spectrum of the β-nitro alcohol (βNA)nitro-triol after 20 hours at 37° C. and pH 12.7 (top.) The depictedchemical shift observed at 8.48 and is indicative of the generation offormaldehyde from the βNA nitro-triol.

FIG. 32—Provides a comparison of ¹H-NMR spectra of formaldehyde producedby the base-catalyzed decomposition of the β-nitro alcoholnitro-ethanol.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method of cross-linking collagen in acollagenous tissue comprising contacting the collagenous tissue with anamount of a formaldehyde-donating compound or a nitrogen oxidecontaining compound effective to cross-link the collagen in thecollagenous tissue. In one embodiment, the collagenous tissue is cornea,sclera, or skin.

In one embodiment of the invention, the collagenous tissue is in asubject. In a preferred embodiment, the collagenous tissue is cornea andthe subject is afflicted with keratoconus or keratectasia.

In various embodiments, the subject is a mammal, for example, a rabbit,a pig, a rat, or a primate, such as a human.

In an embodiment, the nitrogen oxide containing compound is sodiumnitrite or potassium nitrite. In certain embodiments, theformaldehyde-donating compound is a nitrogen oxide containing compound(e.g., a nitro alcohol, such as a β-nitro alcohol.) In anotherembodiment, the nitrogen oxide containing compound is a β-nitro alcohol.In certain embodiments, the formaldehyde-donating compound is a β-nitroalcohol. The β-nitro alcohol may be 2-nitroethanol, 2-nitro-1-propanol,2-nitro-1-pentanol, 3-nitro-2-pentanol,2-hydroxymethyl-2-nitro-1,3-propanediol,2-methyl-2-nitro-1,3-propanediol and/or 2-bromo-2-nitro-1,3-propanediol.

In one embodiment, the formaldehyde-donating compound (e.g., a nitrogenoxide containing compound, such as a βNA) is in an aqueous solutionhaving a pH value of 3 to 8. In a specific embodiment, the aqueoussolution has a pH value of 7.4. In another embodiment, the aqueoussolution has a pH value of 5.0. In yet another embodiment, the aqueoussolution has an alkaline pH. In still other embodiments, the aqueoussolution has an acidic pH (e.g., pH less than about 7) or a weaklyacidic pH (e.g., a pH between about 5 and 7.) By pH value of 3 to 8, itis meant that all hundredth, tenth and integer unit amounts within therange are specifically disclosed as part of the invention. Thus, 3.01,3.02 . . . 7.98, 7.99; 3.1, 3.2 . . . 7.8, 7.9; and 4, 5 . . . 6, 7 pHvalues are included as embodiments of this invention.

Formulating the compounds of the present invention (e.g., one or moreformaldehyde-donating compounds, such as β-nitro alcohols) in an acidicor weakly acidic media (e.g., an aqueous pharmaceutical composition) maybe used as a means to modulate (e.g., improve or otherwise increase) thestability of such compounds or to otherwise render such compoundsresistant to degradation upon storage for extended periods of time. Forexample, formulating the compounds (e.g., a βNA) in an acidic or weaklyacidic media or pharmaceutical composition may render such compoundsstable for at least about one, two, three, four, six, nine, twelve,sixteen, eighteen, twenty four, thirty, thirty six, forty two, fortyeight months or longer, when stored at room temperature and standardhumidity or when stored under accelerated storage conditions.

In one embodiment, the formaldehyde-donating compounds or nitrogen oxidecontaining compounds (e.g., a nitro alcohol) is in an aqueous solutionof sodium phosphate, potassium phosphate, dextran, sodium chloride,potassium chloride, calcium chloride dehydrate, magnesium chloridehexahydrate, sodium acetate trihydrate, sodium citrate dehydrate, andbalance water. In one embodiment, the nitrogen oxide containing compoundis in an aqueous solution of 0 percent to 20 percent sodium chloride byweight/volume, 0 percent to 20 percent potassium chloride byweight/volume, 0 percent to 20 percent sodium phosphate byweight/volume, 0 percent to 20 percent potassium phosphate byweight/volume, 0 to 20 percent dextran by weight/volume, 0 percent to 10percent calcium chloride dihydrate by weight/volume, 0 percent to 10percent magnesium chloride hexahydrate by weight/volume, 0 percent to 10percent sodium acetate trihydrate by weight/volume, 0 percent to 10percent sodium citrate dihydrate by weight/volume, 0 percent to 3percent sodium hydroxide by weight/volume, 0 percent to 3 percenthydrochloric acid by weight/volume, and 0 to 90 percent deionized byweight/volume, and balance deionized, distilled water. In a specificembodiment, the nitrogen oxide containing compound is in an aqueoussolution of 0.50 percent to 1.00 percent sodium chloride byweight/volume, 0.01 percent to 1.00 percent potassium chloride byweight/volume, 0 percent to 10 percent calcium chloride dihydrate byweight/volume, 0 percent to 10 percent magnesium chloride hexahydrate byweight/volume, 0 percent to 10 percent sodium acetate trihydrate byweight/volume, 0 percent to 10 percent sodium citrate dihydrate byweight/volume, 0 percent to 1 percent sodium hydroxide by weight/volume,0 percent to 1 percent hydrochloric acid by weight/volume, and 0 to 90percent deionized, distilled water by weight/volume. In a specificembodiment, the nitrogen oxide containing compound is in an aqueoussolution of 0.64% sodium chloride by weight/volume, 0.075% potassiumchloride by weight/volume, 0.048% calcium chloride dihydrate byweight/volume, 0.03% magnesium chloride hexahydrate by weight/volume,0.39% sodium acetate trihydrate by weight/volume, 0.17% sodium citratedehydrate by weight/volume, and balance deionized, distilled water.Examples of units of a compound in solution by weight/volume are mg/ml,g/100 ml, and kg/L. By percent of a compound in solution, it is meantthat all hundredth, tenth and integer percentages within the range arespecifically disclosed as part of the invention. Thus, 0.01, 0.02 . . .99.98, 99.99; 0.1, 0.2 . . . 99.8, 99.9; and 1, 2 . . . 98, 99percentages are included as embodiments of this invention.

In one embodiment of this invention the formaldehyde-donating compoundsor nitrogen oxide-containing compound is in a solution or solidcomprising a transporter. In a further embodiment of this invention thetransporter is taurine.

In various embodiments, the formaldehyde-donating compound or nitrogenoxide containing compound is in an aqueous solution that can beadministered to skin as a spray, low-viscosity aqueous liquid, alcoholicliquid, mist, aerosol, lotion, gel, cream, ointment, foam, paste,unguent, emulsion, liposomal suspension, colloid, cosmetic, foundation,moisturizer, sun-blocking agent, or combination thereof. In otherembodiment, the aqueous solution may further comprise an antibiotic. Inother embodiments, the aqueous solution may further comprise afragrance. In one embodiment, the aqueous solution is suitable foradministering to skin to prevent wrinkling.

The formaldehyde-donating compounds and the nitrogen oxide-containingcompounds disclosed herein (e.g., βNAs) may be administered to a subjectin need thereof through various routes of administration, which includebut are not limited to ophthalmic, oral, parenteral and topical routesof administration. Such formaldehyde-donating compounds or nitrogenoxide-containing compounds (e.g., βNAs) may be administered alone, butare preferably administered as a composition formulated with one or morepharmaceutically acceptable carriers (e.g., as a topically- orophthalmically-administered solution, suspension, lotion, cream,ointment, hydrogel or gel.)

As used herein, the phrase “pharmaceutically acceptable carrier” refersto a vehicle which is suitable for administration to a subject (e.g., amammal). Preferably the formaldehyde-donating compounds and nitrogenoxide-containing compounds disclosed herein are formulated such thatthey are either soluble or suspended in the pharmaceutically acceptablecarrier. In one embodiment the compositions of the present invention maybe formulated by combining one or more formaldehyde-donating compoundsor nitrogen oxide-containing compounds with one or more pharmaceuticallyacceptable carriers, which may be administered to a subject in a varietyof dosage forms (e.g., tablets, powders, lozenges, syrups, topical andinjectable solutions, suspensions, gels, creams, lotions and the like).In a preferred embodiment, such pharmaceutically acceptable carriers aresuitable for topical, ophthalmic or mucosal administration.

Suitable pharmaceutically acceptable carriers will be readily apparentto those of ordinary skill in the art and the selection of an acceptablecarrier should be optimized based on the selected nitrogenoxide-containing compound and the contemplated route of administration.Pharmaceutically acceptable carriers include, but are not limited to,hydroxypropyl cellulose, starch, pregelatinized starch, gelatin,sucrose, acacia, alginic acid, sodium alginate, guar gum, ethylcellulose, carboxymethylcellulose sodium, carboxymethylcellulosecalcium, polyvinylpyrrolidone, methylcellulose, hydroxyproplymethylcellulose, microcrystalline cellulose, polyethylene glycol,powdered cellulose, glucose, croscarmellose sodium, crospovidone,polacrilin potassium, sodium starch glycolate, tragacanth, calciumcarbonate, dibasic calcium phosphate, tribasic calcium phosphate,kaolin, mannitol, talc, cellulose acetate phthalate, polyethylenephthalate, shellac, titanium dioxide, carnauba wax, microcrystallinewax, calcium stearate, magnesium stearate, castor oil, mineral oil,light mineral oil, glycerin, sorbitol, mannitol, stearic acid, sodiumlauryl sulfate, hydrogenated vegetable oil (e.g., peanut, cottonseed,sunflower, sesame, olive, corn, soybean), zinc stearate, ethyl oleate,ethyl laurate, agar, calcium silicate, magnesium silicate, silicondioxide, colloidal silicon dioxide, calcium chloride, calcium sulfate,silica gel, castor oil, diethyl phthalate, glyercin, mono- anddi-acetylated monoglycerides, propylene, glycol, triacetin, alamic acid,aluminum monostearate, bentonite, bentonite magma, carboxyvinylpolymers, CARBOPOL 934, 940 or 941, carboxymethylcellulose sodium 12,carrageenan, hydroxyethyl cellulose, magnesium aluminum silicate,pectin, polyvinyl alcohol, povidine, sodium alginate, tragacanth,xanthan gum, and silicones.

The compositions of the present invention may further be formulated withadditional pharmaceutical excipients. Suitable excipients may include,but not be limited to inert solid diluents, fillers, sterile aqueoussolutions, organic solvents, thickeners, suspending agents, emulsifiers,buffers, pH modifiers, surfactants, foaming agents, cosmetic adjuvants,anti-oxidants, fragrances, viscosifiers and the like. In one particularembodiment, the pharmaceutical compositions of the present inventioninclude one or more moisturizers (e.g., vitamin A, vitamin E and aloe).The methods of preparing the pharmaceutical compositions of the presentinvention and selection of pharmaceutically acceptable carriers andexcipients are described in detail in, for example, L. William,Remington: The Science and Practice of Pharmacy. 20th ed. MackPublishing Company. Easton, Pa., (2000), the entire contents of whichare incorporated herein by reference.

In a preferred embodiment, the pharmaceutically acceptable carriers andexcipients of the present invention are present in amounts which willnot destroy or alter the activity of the formaldehyde-donating compoundsor nitrogen oxide-containing compounds (e.g., βNAs) present in thecomposition. Preferably, such formaldehyde-donating compounds andnitrogen oxide-containing compounds are present in the compositions ofthe present invention in an amount, based upon the total volume of thecompositions of about 0.025% (w/v), about 0.05% (w/v), about 0.1% (w/v),about 0.2% (w/v), about 0.25% (w/v), about 0.5% (w/v), about 1.0% (w/v),about 1.5% (w/v), about 2.0% (w/v), about 2.5% (w/v), about 5.0% (w/v),about 10.0% (w/v), about 15.0% (w/v), about 20.0% (w/v), about 25.0%(w/v) or more.

In certain embodiments, the formaldehyde-donating compounds (e.g., βNAs)and/or catalysts disclosed herein are formulated in one or morecompositions or suitable carriers that exhibit an extended, controlledand/or delayed release of the one or more constituent compounds (e.g.,βNAs and/or catalysts) that comprise such compositions or carriers. Insome embodiments, the rate at which constituent compounds (e.g., βNAs)are released from the compositions and carriers disclosed herein (e.g.,suspensions, solutions, hydrogels, creams and ointments) may bemanipulated by altering the viscosity or rheology of such compositions.For example, by increasing the viscosity of such compositions, the rateat which the constituent compounds (e.g., βNAs) are released from suchcomposition can be slowed, delayed or otherwise extended. In certainembodiments such compositions are capable of releasing one or more ofthe constituent compounds over the course of at least about 1 hour, 2hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 18 hours,1 day, 36 hours, 2 days, 3 days, 4 days, 1 week or more.

Exemplary excipients that may be included in the compositions andcarriers disclosed herein to increase the viscosity of such compositionsor to impart desired release characteristics may include, but not belimited to, carboxy methylcellulose, hydroxypropyl methylcellulose,hydroxyl ethylcellulose, glycerin, hyaluronidase, polyvinyl pyrrolidone,polyethylene glycol, dextran, hyaluronic acid, carbomer 940 (polyacrylicacid), mannitol, carboxypolymethylene, CARBOPOL, polyvinyl alcohols,carboxy vinyl polymers and other similar agents, as disclosed forexample in L. William, Remington: The Science and Practice of Pharmacy.20th ed. Mack Publishing Company. Easton, Pa., (2000).

The pharmaceutical compositions prepared in accordance with theteachings provided herein are preferably stable. In one embodiment, thepharmaceutical compositions remain stable for at least about 90 daysupon storage at room temperature. In one embodiment, the pharmaceuticalcompositions remain stable for at least 180 days upon storage at roomtemperature. In one embodiment, the pharmaceutical compositions remainstable for at least twelve months upon storage at room temperature. Inone embodiment, the pharmaceutical compositions remain stable for atleast about 2 years (e.g., about three years, four years or more) uponstorage at 37° C.

In certain embodiments the substantial absence of a precipitate,cloudiness and/or other particulate matter (e.g., following storage at37° C. for at least about six, twelve, eighteen, twenty four, thirtysix, forty eight months or longer) in the composition may be indicativeof its stability. In some embodiments the pharmaceutical compositions(e.g., weakly acidic aqueous solutions) disclosed herein are resistantto the development of precipitates or other particulate matter. Forexample, when stored for extended periods (e.g., about one, two, three,six, twelve, eighteen, twenty-four, thirty-six months, or more at roomtemperature), upon visual inspection of the pharmaceutical compositionsdisclosed herein such compositions do not demonstrate phase separationor the development of a precipitate.

In various embodiments, the tonicity of the aqueous solution is from 1milli-osmoles to 100 osmoles.

In various embodiments, the contacting of the formaldehyde-donatingcompound or nitrogen oxide containing compound to the collagenous tissueis performed by intermittent administration of the formaldehyde-donatingcompound or nitrogen oxide containing compound to the collagenous tissuefor a duration of time effective to cross-link collagen. In variousembodiments, the solution is administered at intervals of one to tentimes per day over a period of one day to one hundred and eighty days.In a specific embodiment, the solution is administered one to four timesper day over a period of forty-two days. By administered one to tentimes per day, it is meant that all integer unit amounts within therange are specifically disclosed as part of the invention. Thus, 2, 3, .. . 8, 9 administrations are included as embodiments of this invention.Similarly, the administration may be over a period of 2 days, 3 days . .. 178 days, or 179 days, and each integer value of days is included asan embodiment of this invention.

In one embodiment of this invention, the nitrogen oxide containingcompound is used in a field in which glutaraldehyde has been used tocross-link collagenous tissue. Examples of such fields include, but arenot limited to, heart valves, bioprostheses, drug matrices, tanningleather, and fixing specimen. Nimni, M. E., “Glutaraldehyde fixationrevisited,” Journal of Long-Term Effects of Medical Implants 2001;11(3&4):151-161; Jayakrishnan, A. and Jameela, S. R., “Review:Glutaraldehyde as a fixative in bioprostheses and drug deliverymatrices,” Biomaterials 1996; 17:471-484.

In one embodiment of this invention, the formaldehyde-donating compoundor nitrogen oxide-containing compound (e.g., a βNA) is prepared as asolution and is administered as a composition selected from the groupconsisting of ophthalmic drops, ophthalmic salve, ophthalmic ointment,ophthalmic spray, subconjunctival injection, or intravitreal injection,contact lens, conjunctival insert, ocular time release insert, andsustained release implant. In a preferred embodiment, the solution isadministered as an ophthalmic drop.

This invention also provides a method of inhibiting loss of structuralintegrity of a collagenous tissue during transplantation relatedtransport comprising contacting the collagenous tissue with an amount ofa formaldehyde-donating compound or a nitrogen oxide containing compoundeffective to inhibit loss of structural integrity of the collagenoustissue. In one embodiment, the collagenous tissue is contacted with theformaldehyde-donating compound or nitrogen oxide-containing compoundbefore removal of the collagenous tissue from the donor subject. Inanother embodiment, the collagenous tissue is incubated during transportfrom the donor subject. In certain embodiments, theformaldehyde-donating compound is a nitrogen oxide containing compound.In certain embodiments, the formaldehyde-donating compound is a βNA. Inone embodiment, the nitrogen oxide containing compound is nitrous acid.In one embodiment, the donor subject is a mammal, for example, a rabbit,a pig, a rat, or a primate. In a specific embodiment, the donor subjectis a human. In another embodiment, the donor subject is a pig. In oneembodiment, the collagenous tissue is a heart valve. In anotherembodiment, the collagenous tissue is skin. In yet another embodiment,the collagenous tissue is cornea. In one embodiment, the contacting isat a temperature greater than 60° C. In another embodiment, thecontacting is at a temperature greater than 62° C.

This invention provides a composition for ophthalmic administrationcomprising a formaldehyde-donating compound or a nitrogen oxidecontaining compound (e.g., a βNA), sodium phosphate, potassiumphosphate, dextran, sodium chloride, potassium chloride, calciumchloride dihydrate, magnesium chloride hexahydrate, sodium acetatetrihydrate, sodium citrate dehydrate, and water.

In one embodiment, the composition for ophthalmic administrationcomprising 0 to 100 percent of a nitrogen oxide containing compound byweight/volume, 0 percent to 20 percent sodium phosphate byweight/volume, 0 percent to 20 percent potassium phosphate byweight/volume, 0 percent to 20 percent dextran by weight/volume, 0percent to 20 percent sodium chloride by weight/volume, 0 percent to 20percent potassium chloride by weight/volume, 0 percent to 10 percentcalcium chloride dihydrate by weight/volume, 0 percent to 10 percentmagnesium chloride hexahydrate by weight/volume, 0 percent to 10 percentsodium acetate trihydrate by weight/volume, 0 percent to 10 percentsodium citrate dihydrate by weight/volume, 0 percent to 3 percent sodiumhydroxide by weight/volume, 0 percent to 3 percent hydrochloric acid byweight/volume, and 0 to 90 percent deionized by weight/volume, balancedeionized, distilled water by weight/volume. In a specific embodiment,the composition for ophthalmic administration comprises 0 to 50 percentof a nitrogen oxide containing compound by weight/volume, 0 percent to20 percent sodium phosphate by weight/volume, 0 percent to 20 percentpotassium phosphate by weight/volume, 0 percent to 20 percent dextran byweight/volume, 0.50 percent to 1.00 percent sodium chloride byweight/volume, 0.01 percent to 1.00 percent potassium chloride byweight/volume, 0 percent to 10 percent calcium chloride dihydrate byweight/volume, 0 percent to 10 percent magnesium chloride hexahydrate byweight/volume, 0 percent to 10 percent sodium acetate trihydrate byweight/volume, 0 percent to 10 percent sodium citrate dihydrate byweight/volume, 0 percent to 1 percent sodium hydroxide by weight/volume,0 percent to 1 percent hydrochloric acid by weight/volume, and 0 to 90percent deionized, distilled water by weight/volume.

In a preferred embodiment, the composition for ophthalmic administrationcomprises a nitrogen oxide containing compound, 0.64% sodium chloride byweight/volume, 0.075% potassium chloride by weight/volume, 0.048%calcium chloride dihydrate by weight/volume, 0.03% magnesium chloridehexahydrate by weight/volume, 0.39% sodium acetate trihydrate byweight/volume, 0.17% sodium citrate dehydrate by weight/volume, andbalance deionized, distilled water.

In one embodiment, the composition has a pH of 3 to 8. In a specificembodiment, the composition has a pH of 7.4. In another embodiment, thecomposition has the tonicity of 1 milli-osmoles to 100 osmoles.

This invention also provides a method of altering the refractive powerof a cornea comprising contacting the cornea with aformaldehyde-donating compound or a nitrogen oxide-containing compound(e.g., a βNA) so as to effect cross-linking of collagen in the corneaand thereby alter the refractive power of the cornea. In one embodimentthe nitrogen oxide-containing compound is a β-nitro alcohol. In anotherembodiment the refractive power of the cornea is increased.

In yet another embodiment the cross-linking effected in the corneacauses a surface contour of the cornea to change shape. In a furtherembodiment the cornea is an isolated cornea. In one embodiment thecornea is a porcine cornea. In another embodiment the cornea is a humancornea. In one embodiment the formaldehyde-donating compound is aβ-nitro alcohol (e.g., 2-nitroethanol.) In another embodiment theβ-nitro alcohol is 2-nitro-1-propanol. In one embodiment the β-nitroalcohol is 3-nitro-2-pentanol. In another embodiment the β-nitro alcoholis 2-nitro-1-pentanol. In one embodiment the β-nitro alcohol is2-hydroxymethyl-2-nitro-1,3-propanediol. In one embodiment the β-nitroalcohol is 2-methyl-2-nitro-1,3-propanediol. In one embodiment theβ-nitro alcohol is 2-bromo-2-nitro-1,3-propanediol. In one embodimentthe β-nitro alcohol is 3-nitro-2-pentanol.

As used herein, the phrases “formaldehyde-donating compounds” and“formaldehyde donor compounds” refer to any compounds that may becharacterized as liberating, releasing or otherwise producing analdehyde species (e.g., a βNA capable of liberating one or moreformaldehyde species upon exposure to a catalyst). For example, aformaldehyde-donating compound may dissociate into or otherwise liberateor release one or more aldehyde species (e.g., one, two, three, four ormore formaldehyde species) upon exposure to certain conditions (e.g.,alkaline conditions) or in the presence of certain catalysts (e.g., oneor more enzymes).

In certain embodiments, the formaldehyde-donating compounds arenitrogen-oxide containing compounds, such as nitro alcohols. Forexample, such nitro alcohol formaldehyde donor compounds may be selectedfrom the group of compounds consisting of2-hydroxymethyl-2-nitro-1,3-propanediol,2-methyl-2-nitro-1,3-propanediol, 2-bromo-2-nitro-1,3-propanediol,2-nitroethanol, 2-nitro-1-propanol, 3-nitro-2-pentanol,2-nitro-1-pentanol and combinations thereof. In other embodiments, theformaldehyde donor compounds are selected from the group consisting ofpyrrolidine/2-Methyl-2-nitro-1,3-propanediol (PYRRINMPD),diethylamine/2-Methyl-2-nitro-1,3-propanediol (DEAINMPD),2-Methyl-2-nitro-1,3-prop-anediol-bis-diethylamine (NMPD-bis-DEA),2-dimethylamino-2-hydroxymethyl-1,3-propanediol (DMTA),dimethylamine-2-Methy 1-2-nitro-1,3-propanediol (DMA-NMPD),2-methyl-2-nitro-1,3-propanediol (NMPD) and combinations thereof. Incertain embodiments, the formaldehyde donor compounds are selected fromthe group of oxazolidines, nitroalcohols, nitroacetals, nitro-olefins,nitroamines, aminoalcohols, hexahydropyrimidines, aminonitroalcohols,nitrones, hydroxylamines, imines and combinations thereof. In certainembodiments, the formaldehyde donor compounds are selected from thosedisclosed in U.S. Patent Application Publication No. 2004/0116647 A1,the teachings of which are incorporated herein by reference in theirentirety.

As used herein, the phrase “nitrogen-oxide containing compound” refersto any chemical compound that contains at least one nitrogen oxidefunctional group. Nitrogen oxide containing compounds include, but arenot limited to, nitrites of the general formula RONO, nitro compounds ofthe general formula RNO₂, nitroso compounds of the general formula RNO,and NONOates of the general formula RR′N—(NO—)—N═O, where R and R′ canbe any organic group, such as an acetal, acid anhydride, alcohol,aldehyde, alkane, cycloalkane, alkene, cycloalkene, alkyl, alkylamine,alkyl halide, alkyne, cycloalkyne, allyl, amide, amine, annulene, arene,aryl halide, arylamine, aryne, carbinolamine, carboxylic acid,dicarboxylic acid, hydrocarbon, imide, imine, lactam, lactone, peroxide,phenol, phenyl, polyamide, polyamine, polycyclic aromatic hydrocarbon,polycyclic hydrocarbon, saccharide, thiol, thioester, or a substitutedgroup where the substituent is any of the aforementioned groups. Incertain embodiments, the nitrogen-oxide containing compound is a βNA(e.g., 2-hydroxymethyl-2-nitro-1,3-propanediol,2-methyl-2-nitro-1,3-propanediol, 2-bromo-2-nitro-1,3-propanediol,2-nitroethanol, 2-nitro-1-propanol, 3-nitro-2-pentanol and/or2-nitro-1-pentanol.) In certain embodiments, the nitrogen-oxidecontaining compound is also a formaldehyde-donating compound. In otherembodiments, the nitrogen-oxide containing compound is not aformaldehyde donating compound.

Nitrite-containing examples include, but are not limited to nitrousacid, sodium nitrite, and isopentyl nitrite. Specific nitrites shown towork in this invention include sodium nitrite and potassium nitrite.Some nitrites can be nitrosating agents as defined herein.

Examples of nitro compounds include, but are not limited tonitroalkanes, nitro alcohols and β-nitro alcohols. Specific nitrocompounds shown to work in this invention include 2-nitroethanol,2-nitro-1-propanol, 2-nitro-1-pentanol,2-hydroxymethyl-2-nitro-1,3-propanediol,2-methyl-2-nitro-1,3-propanediol, 2-bromo-2-nitro-1,3-propanediol and3-nitro-2-pentanol.

Examples of nitroso compounds include, but are not limited to2-methyl-2-nitrosopropane and methanamine. Some nitroso compounds can benitrosating agents as defined herein.

Examples of NONOates include, but are not limited to, diethylamineNONOate and spermine NONOate.

As used herein, “nitrosating agent” refers to any chemical compound thatcan impart an NO group onto another molecule. The compound can be eitheran ionic or covalent compound, such as a salt or an ester of nitrousacid. Nitrites of the alkali and alkaline earth metals may besynthesized by reacting a mixture of nitric oxide and nitrogen dioxidewith the corresponding metal hydroxide solution, as well as through thedecomposition of the corresponding nitrate. Nitrites are also availablethrough the reduction of the corresponding nitrates. In one embodiment,“nitrosating agent” refers to, but is not limited to, the groupconsisting of nitrous acid (as nitrosonium ion and/or dinitrogentrioxide); nitrosyl halides of the formula HalNO where Hal is fluorine,chlorine, bromine, iodine, or astatine; nitrosonium salts; alkylnitrites; N-Nitrososulfonamides of the formula RSO₂N(NO)R′, where R andR′ can be any organic substituent, such as an acetal, acid anhydride,alcohol, aldehyde, alkane, cycloalkane, alkene, cycloalkene, alkyl,alkylamine, alkyl halide, alkyne, cycloalkyne, allyl, amide, amine,annulene, arene, aryl halide, arylamine, aryne, carbinolamine,carboxylic acid, dicarboxylic acid, ether, hydrocarbon, imide, imine,ketone, lactam, lactone, peroxide, phenol, phenyl, polyamide, polyamine,polycyclic aromatic hydrocarbon, polycyclic hydrocarbon, saccharide,thiol, or thioester; tetranitromethane C(NO₂)₄; inorganic nitrates;nitrite with carbonyl group catalysts; nitrosyl carboxylates (acylnitrites) of the formula RCOONO, where R has the same definition aspreviously disclosed; Fremy's salt K₂[(SO₃)₂NO]; and sulfur-nitrosocompounds such as thionyl chloronitrite SOCIONO and thionyl dinitriteSO(ONO)₂. In a specific embodiment, “nitrosating agent” refers to thegroup consisting of sodium nitrite and potassium nitrite.

As used herein, “collagenous tissue” refers to any bodily tissue thatcontains the protein collagen, such as skin, blood vessels, heart valve,tendons, bone, cartilage, tendonous tissue, and eye tissues such as thecornea, sclera, and retina. In certain embodiments, the collagenoustissue is human collagenous tissue (e.g., a human cornea). In certainembodiments, the collagenous tissue is mammal collagenous tissue (e.g.,a mammal's corneal, scleral and/or retinal tissue.)

As used herein, “corneoscleral disorder” is any disease, condition, orabnormality of the cornea and/or scleral tissue of the eye involving aloss of stiffness and/or contour changes of the eye. Thus, thecorneoscleral disorder may be keratoconus, keratectasia, progressivemyopia, or glaucoma.

As used herein, the term “transporter” refers to any compound thatallows passage of a nitrogen oxide containing compound into the stroma.An example of a transporter includes, but is not limited to, taurine.

As used herein, the term “tonicity” refers to the ability of a solutionto cause water movement. Tonicity is measured in osmoles, which isdefined by the number of moles of a chemical compound that contribute toa solution's osmotic pressure. In various embodiments of this invention,the solution has a tonicity of between about 1 milli-osmoles and 100osmoles. By tonicity of between about 1 milli-osmoles and 100 osmoles,it is meant that all hundredth, tenth and integer unit amounts withinthe range are specifically disclosed as part of the invention. Thus,1.01, 1.02 . . . 99.98, 99.99; 1.1, 1.2 . . . 99.8, 99.9; and 2, 3 . . .98, 99 osmolar values are included as embodiments of this invention.

As used herein, the term “catalyst” refers to a substance (e.g., anenzyme or DNA) or a condition (e.g., alkaline conditions) thatinitiates, activates or otherwise promotes a chemical reaction. Forexample, the term “catalyst” is used herein to describe compounds and/orconditions that are capable of driving or otherwise promoting thereverse Henry Reaction. Similarly, the term “catalyst” is used herein todescribe compounds and/or conditions that are capable of driving orotherwise promoting the forward Henry Reaction. In some embodiments, thecatalysts promote or otherwise drive the release of one or moreformaldehyde species from a formaldehyde-donating compound.

It should be noted that while in certain embodiments, the catalystsdisclosed herein promote or otherwise catalyze, for example, the forwardHenry Reaction, the observed effect or outcome of such promotion mayeither be an increase or a decrease in the cross-linking of a substratesuch as collagen, chitosan or poly(allylamine) (PAA.) For example,certain catalysts that promote the forward Henry Reaction (e.g.,enzymes) may result in a reduction or deceleration in the rate or degreeof cross-linking of collagen in collagenous tissue.

Exemplary classes of catalysts disclosed herein may include bases (e.g.,aqueous solutions of NaHCO₃, KOH, NaOH, Ca(OH)₂, Na₂CO₃, KF, Bu₃P,MeONa/MeOH, Et₃N), enzymes (e.g., hydroxynitrile lyases,transglutaminases and hydrolases) and double stranded DNA (e.g., salmonsperm DNA).

Transglutaminase enzymes suitable as a catalyst may include, for exampleprotein-glutamine gamma-glutamyltransferases. Hydrolase enzymes suitableas a catalyst may include, for example D-Aminoacylase.

Other catalysts that may be used to initiate, activate or otherwisepromote the forward or reverse Henry Reaction include metal based chiralcatalysts, organocatalysts and tetraaminophosphonium salt. For example,one or more of rare earth metal oxides containing lanthanum, dinuclearzinc catalysts, CU-Bis(oxazoline) catalysts, dual Lewis acid/aminechiral amino alcohol ligands, diethyl zinc triggered reactions,ketoamino cobalt complexes, metal complexes on solid support and lithiumaluminum hydrides may be used in accordance with the teachings of thepresent inventions to promote the forward or reverse Henry Reaction.Similarly, organocatalysts such as guanidine-derived organocatalysts,enatiopure guanidine catalysts, guanidine-based bifunctional catalysts,cinchona alkaloid derived organocatalysts and silyl nitronates may beused in accordance with the teachings of the present invention topromote the forward or reverse Henry Reaction.

Catalysts (e.g., bases, DNA and/or enzymes) that promote the reverseHenry Reaction may accelerate the cross-linking of a substrate (e.g.,collagen) by the formaldehyde-donating compound (e.g., one or more βNAs)by at least about two-, three-, four-, five-, six-, seven-, eight-,nine-, ten-, twelve-, fifteen-, eighteen-, twenty-, twenty five-,thirty-, fifty, one hundred-fold, or more relative to the rate at whichthe formaldehyde-donating compound (e.g., βNA) cross-links suchsubstrate (e.g., collagen or PAA) in the absence of such catalysts.Certain catalysts (e.g., the enzyme hydroxynitrile lyase) that promotethe forward Henry Reaction may decelerate the rate at which theformaldehyde-donating compounds (e.g., βNAs) cross-link a substrate(e.g., collagen in a collagenous tissue) by at least about two-, three-,four-, five-, six-, seven-, eight-, nine-, ten-, twelve-, fifteen-,eighteen-, twenty-, twenty five-, thirty-, fifty, one hundred-fold, ormore relative to the rate at which the formaldehyde-donating compounds(e.g., βNAs) cross-links such substrate (e.g., collagen) in the absenceof such catalysts.

As used herein, the terms “contact” and “contacting” refer to bringingtwo or more substances or reactants (e.g., a formaldehyde-donatingcompound such as a βNA and a catalyst or a βNA and a collagenous tissue)sufficiently close to each other such that the two or more substances orreactants interact or react (e.g., chemically or biologically) with oneanother.

It is to be understood that the invention is not limited in itsapplication to the details set forth in the description or asexemplified. The invention encompasses other embodiments and is capableof being practiced or carried out in various ways. Also, it is to beunderstood that the phraseology and terminology employed herein is forthe purpose of description and should not be regarded as limiting.

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate themethods and compositions of the invention and are not intended to limitthe same.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or theentire group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention encompasses all variations, combinations, and permutationsin which one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. Where elements are presented as lists, (e.g., in Markush group orsimilar format) it is to be understood that each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should be understood that, in general, where the invention, oraspects of the invention, is/are referred to as comprising particularelements, features, etc., certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements, features, etc. For purposes of simplicity those embodimentshave not in every case been specifically set forth in so many wordsherein. It should also be understood that any embodiment or aspect ofthe invention can be explicitly excluded from the claims, regardless ofwhether the specific exclusion is recited in the specification. Thepublications and other reference materials referenced herein to describethe background of the invention and to provide additional detailregarding its practice are hereby incorporated by reference.

EXAMPLES Biaxial Mechanical Tests

Biaxial mechanical properties of the collagen gels were examined byperforming equibiaxial load controlled experiments after 72 hours ofculture for pretreated and 10 days of culture for post-treated collagengels. To perform the planar biaxial mechanical test, the loading framewas first removed and the gels were floated to the top of their Petridish using standard media (DMEM with 10% FBS and antibiotics) at 37° C.Gels were loaded by applying equal weights on all sides of the gelsimultaneously (equibiaxial loading). First, gels were preconditioned bycyclically applying and removing 1 gram weights on all sides.Preconditioning refers to a standard phenomenon in soft biologic tissueswhere the response to an applied load differs with the first fewapplications but eventually stabilizes as the load is repeatedly appliedand removed. In these gels we have found that five cycles are sufficientto obtain a stable response, so five cycles of preconditioning wereapplied to all gels. One prominent feature of the preconditioningresponse in these gels is that the gels undergo some permanentdeformation, not relaxing back to their initial dimensions even whenfully unloaded.

The magnitude of the permanent deformation associated withpreconditioning was quantified by computing the associated strain (E^(P)₁₁, E^(P) ₂₂) using the unloaded configuration of the gel at thebeginning of the mechanical test as the reference state and the new,stable unloaded state achieved at the end of preconditioning as thedeformed state. After preconditioning, calibrated weights from 0 to 1000mg are applied equibiaxially in 100 mg increments. Images of the gelsurface are taken 30 seconds after each load application or removal, andthe strains associated with each load were computed relative to thepreconditioned unloaded reference state at the beginning of the loadingrun.

A fibroblast-populated collagen gel model system was designedspecifically for biaxial loading as seen in FIG. 4. Gel is poured into asquare mold and allowed to polymerize into porous holding bars. Duringdevelopment in the incubator, isotonic uniaxial or biaxial loadingconditions can be applied using weights. Unloaded gels contract to 30%of initial area by 72 hours (open symbols, n=3); contraction decreaseswith load (gray lines) until just detectable at 200 mg per side (closedsymbols, n=3) of 4×4 cm squares.

Collagen Fiber Orientation

Collagen fiber structure was evaluated using confocal reflectancemicroscopy in gels fixed after mechanical testing. Gels were fixed in3.7% formaldehyde in PBS for 24 hours and stored in PBS with 1% sodiumazide at 4° C. Collagen was imaged using a 60× oil immersion objectiveand an argon laser (488 nm) on an Olympus Fluoview 1X70 confocalmicroscope. Digital images (1024×1024 pixel resolution) were thenanalyzed for structural information using a gradient detectionalgorithm. Because collagen is birefringent and confocal microscopy usesa polarized laser, an additional correction was made to remove theresulting bias in the apparent collagen fiber distributions. Fourdifferent fields in the central region of each gel were imaged with thevertical image axis aligned with the x₁ direction, then the gel wasrotated 90° clockwise and four more fields in the central region wereimaged. The collection of eight orientation histograms for each gel wasthen fitted with a sum of two distributions: a “fiber” distributionassumed to rotate with the gel and an “error” distribution assumed toremain stationary.

In the least squares fitting procedure, both distributions were modeledas cosine functions of the form A+B cos(wθ−φ). For the errordistribution, all four parameters in this function were allowed to vary,while the frequency ω of the fiber distribution was fixed at 2 toreflect the natural periodicity of fiber distributions (i.e., a fiberoriented at 0° also can be said to be oriented at 180°, 360°, 540°,etc.).

Biochemical Analysis of Collagen Cross-Linking by SDS-PACE

Samples of pepsin-solubilized, bovine skin collagen type I (Purecol, 3mg/ml, Inamed Corporation, Fremont, Calif., USA) dissolved in 0.012N HClwere spiked with dH₂O containing 1M NaNO₂ or NaCl concentration toobtain final concentrations of 0 mM, 10 mM, 100 mM NaNO₂, and 100 mMNaCl. The addition of the spiking solution resulted in an increase in pHfrom 2.5 to 4.0 for the highest nitrite concentration. These conditionswere identical to those used in the preincubation treatment of collagenfor biomechanical testing. Following a 24 hour incubation (4° C.), analiquot from each sample was added into SDS-PAGE sample buffer (Biorad)in a ratio of 1:2 (sample:sample buffer), reduced by heating at 100° C.for 5 minutes with dithiothreitol (DTT), and analyzed by SDS-PAGE. Theseparating gel was 5% acrylamide and stacking gel 4%. Equivalentquantities of protein (6 μg per lane) were loaded onto separate wells.The samples were run at 25 volts for approximately 3.5 hours, which wasthe time necessary for the dye front to leave the bottom of the gel. Thegels were then stained with Coomasie blue solution for no less than 3hours. Following destaining, the gels were digitally photographed fordensitometric analysis, which was carried out using the Un-Scan-itsoftware program version 4.1 (Silk Scientific Corp).

Collagen Treated by Nitrite

Example of Resistance of Nitrite-Treated Collagen to Digestion

FIG. 1 shows a quantitative protein assay of nitrite-treated collagenfollowing digestion by collagenase. Insoluble type I collagen wasincubated in 100 mM sodium nitrite or sodium chloride solutions for 8weeks. The samples were then digested with bacterial collagenase(Sigma). Protein assay (Biorad) of solubilized protein in thesupernatant fraction shows control collagen was digested more readilythan nitrite-treated collagen at 1, 3, and 5 hours. At 7 and 12 hours,the solubilized fractions were acid hydrolyzed and quantitated by aminoacid analysis. The results from the amino acid analysis were inagreement with these findings. Each point in FIG. 1 represents theaverage of two independent determinations.

Example of Post-Treated Type I Collagen at pH 7.4 Collagen Cross-Linkingis Induced by Reaction with NaNO₂

The results of these studies indicate by SDS-PAGE that collagencross-linking is induced by reaction with NaNO₂. FIG. 2(A) illustrates aSDS-PAGE (5% separating gel stained with Coomasie blue) of type Icollagen (Purecol, Inamed Corp) following a 7 day incubation (1 mg/ml,pH 7.4, 4° C.) with 0, 10, 100 mM NaNO₂, and 100 mM NaCl. At 100 mMNaNO₂ cross-linking of collagen primary strands is indicated by the darkband at the top of the gel. These proteins were unable to enter even thestacking gel. This high molecular weight band has formed in conjunctionwith lesser amounts of lower molecular weight proteins (i.e. α-singlechain, β-dimer, and γ-trimer bands) indicating the formation ofcross-linked collagen strands by reaction with nitrite. No differencesare observed between the unreacted control, 10 mM NaNO₂, and the 100 mMNaCl control. Standard ladder proteins are seen on the right. FIG. 2(B)shows a densitometric analysis of the FIG. 2(A) gel. The digital imagefrom the top panel was scanned using Un-Scan-It software (version 4.1).A marked diminution of alpha, beta, and gamma bands in the high nitritetreated samples is noted with a sharp increase in protein unable toenter the gel. FIG. 2(C) shows biomechanical testing on fibroblastpopulated collagen type I gels.

Biomechanical Stiffening Changes Induced in Collagenous Tissues byCross-Linking by Nitrite

The impact of sodium nitrite on vertical deformation of uniaxiallyconstrained gels was measured during each phase of the experiment. FIG.5(A) shows pretreatment at pH 4.0 for 24 hours with 100 mM sodiumnitrite and post-treatment at pH 7.4 for seven days with 100 mM sodiumnitrite both reduced vertical remodeling. *p<0.05 versus control. FIG.5(B) shows pretreatment at pH 4.0 for 24 hours with 100 mM sodiumnitrite and post-treatment at pH 7.4 for seven days with 100 mM sodiumnitrite both reduced vertical deformation associated withpreconditioning. FIG. 5(C) shows pretreatment at pH 4.0 for 24 hours andpost-treatment at pH 7.4 for seven days with 100 mM sodium nitrite bothreduced vertical deformation at 1 g equibiaxial load.

Example of Nitrite Concentration in Aqueous Humor of the Eye at pH 7.4

Adult porcine eyes were obtained within 12 hours of sacrifice andsubmerged in solutions of 0, 10, 100 mM NaNO₂ and 100 mM NaCl bufferedwith 50 mM NaH₂PO₄/Na₂HPO₄ (pH 7.4) following bubbling with 99% Argon/1%O₂ in order to limit the amount of potential nitrite auto-oxidationcaused by oxygen. Penicillin/Streptomycin was added (10 μl/ml) toprevent bacterial overgrowth. After 24 hours of incubation at 4° C., theaqueous humor was sampled for nitrite concentration using a modificationof the Greiss colorimetric assay. No nitrite was detected in either thebuffer control or the 100 mM NaCl control. Nitrite concentration in theaqueous humor was 35.5% of the incubation fluid in the 10 mM NaNO₂sample and 62.8% in the 100 mM NaNO₂ sample. These results suggest thatthere is a concentration dependent penetration of nitrite through thecornea as evidenced by FIG. 6.

Example of Nitrite Treated Eye Tissue at pH 5.0

Adult porcine eyes were obtained within 12 hours of sacrifice andsubmerged in a solution of either 200 mM NaNO₂ or 200 mM NaCl bufferedwith 200 mM NaH₂PO₄/Na₂HPO₄ (pH 5.0). Penicillin/Streptomycin was added(10 μl/ml) to prevent bacterial overgrowth. After 30 hours of incubationat 37° C., the corneas were excised at the limbus and photographed.Dramatic contour changes in the corneas were observed in the samplescross-linked with NaNO₂ as compared with the NaCl control asdemonstrated by FIG. 7. FIG. 8 demonstrates the dramatic contour changesare seen in sclera samples cross-linked with NaNO₂ as compared with theNaCl control.

After 30 and 80 hours of incubation at 37° C., the corneas were excisedat the limbus and subjected to shrinkage temperature (thermaldenaturation) analysis. The intact cornea was heated in 2° C. incrementsevery 5 minutes using a digitally controlled water bath. The maximaldimensions in the long and short axis were measured using a micrometerunder visualization with an operating microscope. The percentage oftissue shrinkage was then estimated by using 2 dimensional areacalculations from the micrometer readings. The onset of tissue shrinkagewas increased by approximately 4° C. following 30 hours of treatment and8° C. following 80 hours of treatment as demonstrated in FIG. 8. Thesefindings provide a general assessment of the degree of collagencross-linking induced by reaction with nitrite.

Developments in Using β-Nitro Alcohol to Cross-Link Collagen

This study uses a combination of thermal denaturation temperatureanalysis, biomechanical testing, and cell culture cytotoxicity in amethod development program aimed at identifying an effective and safemeans of cross-linking collagenous tissues in vivo. These studies arefollowed by trials of topical cross-linking in the living rabbit eye. Asa group, these are translational studies which are designed to leaddirectly to the initiation of a human clinical phase I trial.

Determining an Effective Way to Cross-Link the Cornea Using NitroTechnology Under Conditions Simulating the Human Cornea (i.e., pH 7.4and 34° C.)

Previous studies indicate that reactions with free nitrite ion undersystemic physiologic conditions of neutral pH and body temperatureinduce collagen cross-linking (Paik, D. C., et al., 2001; Paik, D. C.,et al., 2006). This has been shown through general measures ofcross-linking such as increased thermal shrinkage temperature andincreased resistance to enzymatic digestion, as well as more specificmeasures such as increased intermolecular covalent bonding (by SDS-PAGE)and the formation of lysine derived di- and tri-functional cross-linksby LC/MS (unpublished). However, such reactions with free nitriterequire high concentrations (i.e., 100-200 mM) and prolonged incubationperiods (7-10 days). Thus, methods involving nitrite and related agentsthat significantly speed the reaction under conditions that simulatecorneal tissue (pH 7.4 and 34° C.) have been developed.

Thermal shrinkage temperature or denaturation temperature (TO is atechnique that has been used for decades in studying collagencross-linking. As collagen fibers are progressively heated, a thresholdtemperature is reached at which point disruption of hydrogen bonding inthe collagen molecule triple helix occurs, resulting in unwinding of thetriple helices. The denaturation of tertiary protein structure resultsin a rapid shrinkage of the tissue of up to 80%. This phenomenon can beexpressed in various ways including temperature of onset of tissueshrinkage, temperature at maximal tissue shrinkage, and temperature at50% of maximal tissue shrinkage, for example. In addition, differentialscanning calorimetry (DSC) is related to thermal shrinkage temperatureanalysis and is widely used to evaluate biomaterial cross-linking.Thermal shrinkage temperature is essentially a simplified version of DSCand can be used effectively to screen the cross-linking efficacy ofcompounds. For the purposes of this study, the onset of shrinkage isconsidered the T_(s) and the temperature at which 50% of maximalshrinkage has occurred is T₅₀. Graphs depicting the relationship betweendegree of shrinkage and temperature indicate differences in degree ofcross-linking between groups. Thus, stabilization of collagen fiberstructures through non-enzymatic cross-linking increases T_(s) and T₅₀.A 5° C. upward shift in T_(s) for the anterior corneal stroma has beenshown for the UVR cross-linking method (Spoerl, E., et al., 2004).

Polyethylene Box for Shrinkage Temperature Analysis

A semi-automated system has been developed which increases theefficiency and accuracy of T_(s) data collection. This system canperform multiple T_(s) assays in succession, allowing rapid screening ofa number of potential cross-linking agents and conditions. The setupconsists of a custom built polyethylene box for shrinkage temperatureanalysis, as shown in FIG. 10, into which tissue samples are placed intoan insert. Inlet and outlet fittings allow for constant circulation ofheated water which is modulated by a digitally controlled water bathequipped with a water pump. The entire box is placed on a digital photoscanner connected to a Windows based computer. Beginning at 50° C. thetemperature is raised at a rate of 1° C./min and serial scans are takenat every degree increase in temperature up to 80° C. Control pig corneasshrink at approximately 63° C. and cross-linked tissue at highertemperatures. As the tissue shrinks, the images are captured by thescanner. The image files are later analyzed for 2-dimensional area usingNIH Image J software and % shrinkage calculations are made usingMicrosoft Excel for graphing purposes. This method obviates the need forremoval of tissue pieces from the water bath with manual micrometermeasurements as described by the UVR group (Spoerl, E., et al., 2004).Thus, the labor involved in acquiring data is lessened and the accuracyusing image analysis is improved.

FIG. 11 illustrates an example of shrinkage temperature change inporcine sclera cross-linked with 2-nitroethanol (2NE). Serial images areshown depicting shrinkage of control samples in the 66° C. image but notthe 2-nitroethanol. At a higher temperature (i.e. 70° C.), the 10 mM2-nitroethanol samples shrink. Finally, at the highest temperature (i.e.74° C.), all the samples have shrunken, including the 100 mM2-nitroethanol samples. This example depicts the concept thatcross-linked collagenous tissues require higher temperatures in order toshrink (or denature).

Using this simple, reproducible assay enables the screening of a numberof compounds for cross-linking efficacy. This is first performed usingporcine eyes because of their low cost. Follow up studies can beperformed using human eyes. Porcine eyes are obtained within 6 hrs ofsacrifice (Hatfield Meat Packing, Inc., Hatfield Pa.). Eyes showingclear corneas and intact epithelia are chosen for study. 10×4 mm stripsare obtained from the central corneal region along the superior-inferioraxis (2 per eye). Each strip is then cross-linked using a nitro agent.The incubation solution is 20%, Dextran (T500) [to prevent tissueswelling] in 0.2 M NaH₂PO₄/Na₂HPO₄ (pH 7.4) with various concentrationsof nitro agent (i.e. 1, 10, and 100 mM). At these concentrations pH iswell maintained over the course of 96 hrs. The samples are then placedin 2 mL microcentrifuge tubes with 1 mL of incubation solution andreacted in a 34° C. water bath (simulating corneal temperature—Girardin,F., et al., “Relationship between corneal temperature and fingertemperature,” Arch. Ophthalmol. 1999; 117:166-169) for 24-96 hrs.Following incubation, the samples are evaluated for degree ofcross-linking using the T_(s) assay as described above.

From the data, T_(s) curves are generated for each compound andcondition studied. Each data point is the average of 3 independentsamples. Differences between groups can be compared for statisticalsignificance using simple t-test analyses comparing T_(s) and T₅₀. Usingthese curves, comparisons are made between compounds regarding theircross-linking efficacy. The targeted change for onset of shrinkagetemperature is 5° C. This is the level of change reported by the UVRgroup for cross-linking. Compounds are sought that can produce thetarget T_(s) (−5° C.) shift at the lowest concentration and shortestpossible incubation times. Ultimately, however, the choice of compound(or compounds) for cross-linking in the living eye is determined basednot only on its cross-linking efficacy but also its cytotoxicity, whichis determined as described herein.

Comparing β-Nitro Alcohols

Different β-nitro alcohols are compared with regard to cross-linkingefficacy. At least 3 of the lower order β-nitro alcohols related to2-nitroethanol (i.e. 2-nitro-propanol, 2-nitro-1-pentanol), which iseffective as a cross-linking compound, are studied. This determines thedifferences in cross-linking efficacy between various β-nitro alcohols.It also provides potential alternatives for in vivo rabbit eyecross-linking in the event that any of these compounds exhibitsignificant cytotoxicity to corneal cells. When a candidate agent isidentified (such as 2-nitroethanol) human eye bank corneas are tested inorder to confirm the effects in human tissue. Because these compoundsare used for cross-linking living corneas over the course of weeks,cross-linking efficacy occurring over many days at low concentrationsare studied. This simulates the in vivo use of serial reagentapplication lasting on the order of weeks. For example, if 1 mM is thehighest non-toxic level for 2-nitroethanol, a time course study isconducted in which tissue samples are reacted over the course of 2-3weeks by replacing the incubation fluid daily with 1 mM 2-nitroethanol.The goal in this regard is to determine the length of duration necessaryfor adequate cross-linking at the non-toxic dose level.

Previous studies using acidified NaNO₂ indicate that cross-linking isenhanced by lowering the pH. This indicates that nitrous acid and thesubsequent formation of nitrosating species such as nitrosonium (+NO)and/or dinitrogen trioxide (N₂O₃) are involved in cross-linking. If thiswere the case, it would not be predicted that the β-nitro alcohols couldcross-link as well. However, 2-nitroethanol does induce cross-linking.Nitrous acid (i.e. NaNO₂ compared at pH 3, 5, and 7.4), isopentylnitrite (a well-known nitrosating agent—Williams, D. L. H., “Reagentseffecting nitrosation. In: Nitrosation Reactions and the Chemistry ofNitric Oxide,” 2004, ed., Elsevier, Chapter 1:1-34; Iglesias, E. andCasado, J., “Mechanisms of hydrolysis and nitrosation reactions of alkylnitrite in various media,” Int. Rev. Phys. Chem. 2002; 21(1):37-74), and2 selected diazeniumdiolate compounds, dipropylenetriamine NONOate(DPTA/NO) (t/2=3 hr) and diethylenetriaamine NONOate (DETA/NO) (t/2-20hr) (Kong, L., et al., “Deamidation of peptides in aerobic nitric oxidesolution by a nitrosative pathway,” Nitric Oxide 2006; 14(2):144-151)are used. By using 4 distinct means for producing nitrosation, it can bedetermined if nitrosation induces collagen cross-linking (as determinedby a shift in T_(s)). Incubations are performed at 100 mMconcentrations. Confirming or rejecting nitrosation as a cross-linkingmechanism provides important information. It is well known thatnitrosation of a primary amine forms an unstable diazo compound whichundergoes dediazoniation (N₂ gas is liberated). In this way nitrosationof the primary ε-amines groups in lysine and hydroxylysine (importantcross-linking sites in collagen) with resultant deamination is aplausible mechanism.

Finally, there are several potential benefits to using free nitrite as atopical stiffening agent. First, nitrite is a physiologic molecule andis well tolerated by cells. Second, based on preliminary studies nitriteappears to penetrate the corneal epithelium. This being said, at neutralpH, the reaction with nitrite is rather slow, requiring several days forcross-linking effects to occur. Thus, whether the rate of reaction withfree nitrite at neutral pH can be increased through use of a catalyst isstudied. Previous studies by Keefer, et al. in the early 1970's showedthat neutral pH nitrite reactions could be enhanced by the addition ofaldehyde catalysts. Keefer, L. K. and Roller, P. P., “N-nitrosation bynitrite ion in neutral basic medium,” Science 1973; 181(4106):1245-1247.Such compounds included pyridoxal (vitamin 56) which should be welltolerated by cells and other aldehyde compounds, which can be added tothe nitrite solution in low concentrations in order to speed thereactions.

Testing that Corneal Cross-Linking Through Nitro Technology IncreasesBiomechanical Stiffness Properties Commensurate with UV/RiboflavinTherapy

It has been shown that the keratoconic cornea displays alterations inelasticity compared to normal corneas, indicating a decreased stiffness.Andreassen, T. T., et al., 1980; Nash, I. S., et al., “Comparison ofmechanical properties of keratoconus and normal corneas,” Exp. Eye Res.1982; 35:413-423; Edmund, C., “Corneal elasticity and ocular rigidity innormal and keratoconic eyes,” Acta Ophthalmol. 1988; 66:134-140. Thus,the ultimate goal of corneal cross-linking is to provide mechanicalstabilization to the cornea by increasing the tissue stiffness. AlthoughT_(s) measurements provide a reasonable assessment of the degree oftissue cross-linking, it is necessary to establish the degree ofbiomechanical strength imparted by nitro technology through mechanicaltesting. Thus, the range of biomechanical stiffness achievable throughthese reactions is established.

Fresh porcine and human cadaver whole corneas are cross-linked usingmethods developed in the first part of Determining an Effective Way toCross-Link the Cornea Using Nitro Technology under Conditions Simulatingthe Human Corea (i.e., pH 7.4 and 34° C.). Based on preliminary studies,this is a β-nitro alcohol compound similar or identical to2-nitroethanol. However, this can also include nitrosating compoundssuch as the diazeniumdiolates if they are determined to be effectivecross-linkers as well as free nitrite with a catalyst. All corneas aretested while fully immersed in hypertonic 30% NaCl (to prevent swelling)with phosphate buffer (˜pH 7.4) at room temperature. By using 30% NaClit has been determined that after 4 hours tissue thickness increases byless than 10%. Using lower concentrations of NaCl results in thicknessincreases greater than 10% and therefore is not suitable for mechanicaltesting. Dissected corneas are attached to a custom biaxial stretchingdevice, shown in FIG. 12, using two metallic hooks per side. Graphitemarkers are placed on the center of the anterior surface with adhesiveand imaged for optical deformation tracking with a charged coupleddevice (CCD) video camera mounted above the specimen. Each specimen issubjected to three loading protocols: uniaxial stretches in thehorizontal (i.e. naso-temporal) and vertical (superior-inferior)directions, and equibiaxial stretch. Each test is performed from a 5 gpreloaded equibiaxial state for 10 cycles, 20 seconds per cycle.Equibiaxial tests are repeated throughout the test to verify therepeatability of the mechanical response. Grashow, J. S., et al.,“Biaxial stress-stretch behavior of the mitral valve anterior leaflet atphysiological strain rates,” Ann. Biomed. Eng. 2006 February;34(2):315-325.

During testing, custom software acquires data simultaneously from theCCD video camera (for marker tracking) and the force transducers,allowing calculation of stress-strain curves for each test. During thelast loading cycle (i.e. #10), the deformation of the markers is used tocalculate the deformation gradient F and the Green strain tensor,E=0.5(F^(T)*F−I), where I is the identity tensor. Specimen thickness iscalculated prior to testing in the unloaded state by averaging at leastfive measurements taken at different locations of the cornea using acaliper. The length of each side of the cornea (i.e., length betweensutures) is measured from images taken from the bottom of the sample.The cross-sectional reference area of the sample is calculated and thefirst Piola-Kirchoff stresses P are obtained by dividing the measuredforces by the reference area in the appropriate direction. Assumingincompressibility (J=det F=1) and plane stress (F₁₃, F₂₃, F₃₁, and F₃₂are equal to zero), Cauchy stresses t are computed for the horizontal(t₁₁) and vertical (t₂₂) directions. No less than 3 samples are studiedfor each treatment group. Since the cornea is an anisotropic tissue(Dupps, W. J. and Wilson, S. E., “Biomechanics and wound healing in thecornea,” Exp. Eye Res. 2006; 83:709-720), using the biaxial testerrather than a uniaxial device provides the advantage of obtainingstress-strain relationships along both the superior-inferior as well asthe nasal-temporal axis. The methods developed and range of stiffnesschanges determined are used to predict the changes that are inducible inthe living rabbit eye. It is also allows comparisons to be drawn withthe UVR published values for biomechanical stiffness changes induced byUVR and other chemical cross-linking agents. Wollensak, G. and Spoerl,E., “Collagen crosslinking of human and porcine sclera,” J. CataractRefract. Surg. 2004; 30:689-695.

Testing that Corneal Cross-Linking Through Nitro Technology is Toleratedby Corneal Cells

A number of aldehyde cross-linking agents have been used previously forin vitro collagen cross-linking. Although effective cross-linkingagents, their in vivo utility is limited by their significantcytotoxicity. The clinical utility of this nitro technology isdetermined both by its efficacy as a cross-linking agent as well as itslevel of cytotoxicity. Therefore, relevant cytotoxic effects usingcompounds determined to be efficient cross-linking agents were examinedand include nitrite and related compounds. The cytotoxic effects oncorneal endothelial cells, keratocytes, and epithelial cells wereexamined. Of these, the effects on endothelial cells are the mostimportant, since these cells are principally responsible for maintainingcorneal transparency through regulation of stromal water balance andlack the ability to regenerate in vivo.

Primary cultures of bovine corneal endothelial cells, keratocytes, andepithelial cells are grown in vitro. For endothelial cells, fresh bovineeyes were obtained. The entire corneal ring was cut out and placed intoa concave container used for the storage of contact lenses. A 0.05%trypsin-0.02% EDTA solution was applied onto the endothelium for 5 minin order to dissociate the cells. After 5 min of digestion, theendothelial cells were mobilized using a glass spatula. Finally, thesolution containing suspended endothelial cells was pipetted andtransferred to a 25 cm² cell culture flask filled with 20 ml DMEMcontaining 10% fetal calf serum which quenches the digestive activity oftrypsin. The primary cultures were then placed in a cell culture oven at37° C. and gassed with 6% carbon dioxide. Cell growth was evaluatedevery other day using an inverted phase contrast microscope (ZeissAxiovert L5). The media was changed every 3-4 days. Confluence withabout 2.5×10⁶ cells per flask is reached after 2-3 weeks. For passaging,the confluent stock cultures were dissociated and detached using a 0.05%trypsin-0.02% EDTA solution. The free floating cells were centrifuged at230 g, again transferred to culture flasks and suspended in the cellculture medium. Passaging is performed every 2^(nd) week at a splitratio of 1:3. Grant, M. B., et al., “Effects of epidermal growth factor,fibroblast growth factor, and transforming growth factor-β on cornealcell chemotaxis,” Investigative Ophthalmology & Visual Science 1992;33(12):3292-3301; Orwin, E. J. and Hubel, A., “In vitro culturecharacteristics of corneal epithelial, endothelial, and keratocyte cellsin a native collagen matrix,” Tissue Engineering 2000; 6(4):307-319;Bednarz, J., et al., “Effect of three different media on serum freeculture of donor corneas and isolated human corneal endothelial cells,”Br. J. Ophthalmol. 2001; 85:1416-1420. The approach for preparingprimary keratocyte cultures is similar to that described for endothelialcells, with the exception being that keratocyte are grown from explantedtissues as described by Wollensak, G., et al., “Keratocyte apoptosisafter corneal collagen cross-linking using riboflavin/UVA treatment,”Cornea 2004; 23(1):43-49. Similarly, the protocol for growing epithelialcells is straightforward and has been described in detail. This includesthe use of a specialized growth medium for corneal epithelial cells.Grant, M. B., et al., 1992, Orwin, E. J. and Hubel, A., 2000. Aftergrowing in 96-well plates and with cells at 80-90% confluence, compoundsof interest were added into the culture medium at a range ofconcentrations (0.001-1%). Following a 48 hrs incubation period, a cellsuspension was obtained by trypsinization and dead cells were stainedusing 0.4% trypan blue for 5 min. The live/dead cells were then countedusing a hemocytometer and the dead cell percent calculated. Three wellswere used for each condition. Based on preliminary experiments with2-nitroethanol, a toxicity threshold occurs (>1 mM) such that below thethreshold all cells are alive and above the threshold all cells aredead. A similar protocol was performed to evaluate the degree ofapoptosis and necrosis, this utilizes annexin V and propidium iodidestaining according to the manufacturer's protocol (Molecular Probes,Inc.).

An abrupt toxicity threshold was observed as was described by the UVRgroup, a table was generated for each compound of interest, indicatingthe concentrations below and above the threshold as either beingnon-toxic (i.e. minus sign −) or toxic (plus sign +) (Tables 1 and 2).Trypan blue and PI staining provide a general indication of cytotoxicityand annexin V staining can detect apoptosis. However, clearly there aremore subtle alterations in cell function that may be pertinent tocytotoxicity. For example, alterations in gene expression and potentialmutagenicity could be occurring at a level below the threshold fornecrosis. These more subtle aspects are reserved for later phases ofdrug development during which time extensive evaluation by highthroughput technology can be conducted. Several toxicity/mutagenicitystudies have already been published. For instance, β-nitro alcohols areknown to have a very low mutagenicity (Conaway, C. C., et al., 1991) andanimal toxicity profiles (Jung, Y. S., et al., 2004). As such, they havebeen proposed for use in animal feeds in order to control food bornepathogens in ruminants and chickens where they exhibit bacteriostaticactivity (Horrocks, S. M., et al., 2007).

TABLE 1 Cytotoxic threshold (using trypan blue) of primary bovinecorneal endothelial cells following 48 hrs exposure (mM) 2- 2-nitro-1-3-nitro-2- Concentration nitroethanol propanol pentanol 10 + + + 7 + + +5 + + + 3 + + + 2 − + − 1 − + − 0.75 − − − 0.5 − − − 0.1 − − − + =positive trypan blue staining = cells dead − = no trypan blue staining =cells alive

TABLE 2 Apoptosis (Annexin V) staining of bovine corneal endothelialcells following a 48 hrs exposure (mM) 2- 2-nitro-1- 3-nitro-2-Concentration nitroethanol propanol pentanol 3 + + + 2 − + − 1 − + −0.75 − − − 0.5 − − − 0.1 − − − + = positive trypan blue staining = cellsdead − = no trypan blue staining = cells alive

As demonstrated herein, the highest tolerated level for 2-nitroethanolon ARPE-19 cells is 1 mM. At this concentration, T_(s) for porcinecornea was shifted 1-2° C. when serially applied over 6 days and T_(s)for porcine sclera was shifted 2-3° C. when serially applied over 10days. Based on these studies, it is believed that serial applicationusing lower doses can produce cross-linking effects similar to thoseinduced at higher concentrations for shorter incubation periods. Thistherapy may produce corneal cross-linking over a relatively long period(2-8 weeks) using a dose of drops that would produce a subtoxic stromaltissue level.

TABLE 3 The cytotoxic threshold compares favorably with other ophthalmicagents Species of origin (corneal Toxic Time of endothelial AssayCompound concentration exposure cell) method citation 2-  3 mM 48 hrsBovine Trypan nitroethanol (0.0273%) primary blue 2-nitro-1-  1 mM 48hrs Bovine Trypan propanol (0.0105%) primary blue 3-nitro-2-  3 mM 48hrs Bovine Trypan pentanol (0.0339%) primary blue Ciprofloxacin  3.02 μM15 min Human Calcein Skelnik (0.0001%) AM 2003 Moxifloxacin  2.49 μM 15min Human Calcein Skelnik (0.0001%) AM 2003 Gatifloxacin  2.66 μM 15 minHuman Calcein Skelnik (0.0001%) AM 2003 Levofloxacin  2.69 μM  1 hrHuman Calcein Skelnik (0.0001%) AM 2003 Benzalkonium (0.0001%)  3 hrsRabbit Electron Green chloride organ micro- 1977 culture scopy Cetyl- 10 μM  3 hrs Rabbit Electron Green pyridinium organ micro- 1977chloride culture scopy Daunorubicin 948 nM  7 days Human Calcein Garweg(0.00005%) immor- AM 2006 talized Mitomycin C 299 μM  7 days HumanCalcein Garweg (0.00001%) immor- AM 2006 talized Azathioprine 220 μM  7days Human Calcein Garweg (0.005%) immor- AM 2006 talized Cyclosporin A 41 μM  7 days Human Calcein Garweg (0.005%) immor- AM 2006 talizedProvidone (0.1%) in 12 hrs Bovine Trypan Naor iodine serum free primaryblue 2001 medium

Testing that Corneal Cross-Linking Through Nitro Technology has Efficacyand is Safe for the Living Eye

Evaluation of the efficacy and safety in the living eye is a crucialprerequisite leading to a phase I human clinical trial. The testingdescribed above provides the necessary background information for use ofthis technology in the living eye. Compound selection, concentration,duration of exposure, toxicity thresholds, and target biomechanicaleffects are all pre-estimated from the biochemical, biomechanical, andcell biology studies described above. This provides a general range ofthe conditions necessary for cross-linking the living rabbit cornea.However, considerations with living eyes are far different from in vitrostudies. As such, much of the experimental protocol particularlyconcerning dosing is determined empirically. The results establish thein vivo efficacy and safety of this technology and dictate thefeasibility of a human phase I clinical trial.

A total of 20 young female New Zealand white rabbits weighing 2.0 to 2.5kg are obtained and maintained in the CUMC animal facility on the 8^(th)floor of the Harkness Eye Institute. After a 1 week acclimatizationperiod, a pretreatment baseline evaluation is performed using 2sophisticated, widely used, non-invasive instruments. One of these isthe Ocular Response Analyzer (ORA) from Reichert, Inc., which is used tomeasure intraocular pressure (IOP) and biomechanical properties such ascorneal hysteresis (CH) and corneal resistance factor (CRF). Pepose, J.S., et al., “Changes in corneal biomechanics and intraocular pressurefollowing LASIK using static, dynamic, and noncontact tonometry,” Am. J.Ophthalmol. 2007; 143:39-47. The other instrument is the ConfoScan4 fromNidek Corp., which is a fully digital ophthalmic confocal microscopesystem. This is used to evaluate corneal thickness (pachymetry, an indexof endothelial cell function) and cell toxicity through evaluation ofcorneal endothelial cells, keratocytes, and epithelial cells. Theadvantage of using the ConfoScan4 is that the system is fully automatedfor cell counts and includes important parameters of endothelial cellhealth. This includes measurements of mean cell density, mean cell area,coefficient of variation in cell size (polymegathism), and thepercentage of hexagonal cell (pleomorphism). Bourne, W. M. and McLaren,J. W., “Clinical responses of the corneal endothelium,” Exp. Eye Res.200478:561-572. In addition, corneal thickness and percentage of viablekeratocytes and epithelial cells are obtained during the sameexamination.

During each evaluation, the animals are sedated using an intramuscularinjection of ketamine hydrochloride 10% (35 mg/kg) and xylazinehydrochloride (5 mg/kg) which is dosed on a by-weight basis. Allmeasurements are obtained with the animals under anesthesia. Afteracquisition of the baseline values of each animal using the ORA andConfoScan4, the treatment process begins. Instillation of the topicalcross-linking solution to the right eye with the left eye as a controlis performed daily for a 2-8 week period. Non-invasive measurements aretaken weekly. Of note, when the drops are applied, the compound israpidly mixed with the tear film and most of the administered drop islost to drainage in the first 15-30 seconds. Since the tear turnoverrate is approximately 16% per minute, most of the compound is expectedto disappear within 10 minutes. Thus, the concentration rapidly declinesto a fraction of the applied dose and the amount of time that theepithelium is exposed to the agent is on the order of seconds. McGee, D.H., et al., “Safety of Moxifloxacin as shown in animal and in vitrostudies,” Survey of Ophthalmology 2005; 50(suppl. 1):S46-S54. Theability of the compound to enter the corneal stroma is an importantconsideration. Preliminary studies indicate that free nitrite is able topass into the stroma and into the aqueous chamber with a concentrationdependent effect. However, these studies were performed on cadavereyeballs over 24 hrs. Thus, it is not possible to positively predict,based on those experiments, what the permeability is in a living rabbiteye. Since compounds such as nitrite and 2-nitroethanol are hydrophilic,they are not expected to be able to pass through the lipid bilayer ofcell membranes by passive diffusion. However, there are severaltransporters present in the corneal epithelium that could potentiallyallow rapid passage of these types of compounds into the stroma.Transporters for various amino acids and compounds such as taurine couldprovide a means for easy entry. Mannermaa, E., et al., “Drug transportin the corneal epithelium and blood-retina barrier: emerging role oftransporters in ocular pharmacokinetics,” Advanced Drug Delivery 2006;58:1136-1163.

Initial studies indicate that a 1 mM concentration of 2-nitroethanol cancross-link corneal tissue and is tolerated by living epithelial cells.In vitro findings are extrapolated to considerations related to theliving eye. An eye drop concentration for testing live rabbit cornea, toachieve local stromal concentration of 1 mM 2-nitroethanol, isdetermined largely on an empirical basis to penetrate through thecorneal epithelium. However, nitrite is known to possess the ability tobind non-covalently with tissue and can accumulate differentially intissues. Bryan, N. S., et al., “Cellular targets and mechanisms ofnitros(yl)ation: an insight into their nature and kinetics in vivo,”PNAS 2004; 101(12):4308-4313. Non-covalent binding is also evidenced bythe in vitro finding that following reactions with nitrite and collagen,complete removal of free nitrite requires extensive dialysis using highconcentration buffers and/or use of detergents such as SDS andguanidine. Thus, introducing the agents into the stroma may allow foraccumulation of reagent locally.

Experiments are conducted using a relatively high dose of agent (i.e.100 mM or 1%) on only 2 rabbits. This is applied through continuousapplication of drops for 5 minutes applied daily. By following thecytotoxic effects using the ConfoScan4 and biomechanical propertiesusing the ORA the drug dosage and duration of exposure is able to beadjusted to compensate for changes in the cytotoxicity and/orbiomechanics. Whether the corneal cells can tolerate the application ofa 100 mM 2-nitroethanol solution applied daily is determined. Theduration of application in these experiments is guided by evaluation ofcell viability. If a negative change is observed in endothelial cellparameters, for example, the concentration or length of continuous dropapplication (i.e. 5 min) is decreased. Dosage modulations in the initialexperiments are performed on a per weekly basis and are dictated by dataobtained from the ConfoScan4. A cytotoxic change is indicated by adecrease in mean cell density and/or mean cell area as well as by anincrease in pleomorphism or polymegathism. The threshold for changingdosing is a 10% or greater negative change in any of the 4 endothelialcell parameters. Simultaneously, data obtained by the ORA during thesame treatment process provides a real time assessment of stiffnesschange that is coupled with cell toxicity parameter from the ConfoScan4.Thus, even prior to obtaining post-mortem biomechanical stiffness data,some inference is made as to whether the treatment results in cornealstiffening. After the total treatment period is complete (i.e. 2-8weeks), the animals are sacrificed by CO₂ narcosis and the eyes removedfor post-mortem mechanical testing. That is, whole excised corneas aremounted in the biaxial material tester and stress-strain curvesgenerated in order to draw comparisons to the ORA stiffness data as wellas published data related to the UVR cross-linking method.

Following this initial experiment, subsequent experiments are performedbased on the observed efficacy and cytotoxicity observed at the initialdosage and duration trial with 2 rabbits. The treatment regimen can thenbe modulated either to increase or decrease the concentration of agent,time of continuous application (i.e. minutes), and duration of treatment(i.e. weeks) to optimize the cross-linking efficacy and minimize thetoxicity. Ultimately, up to 6 groups of 3 rabbits per group (i.e. 3rabbits at a time) for a total of 18 rabbits are used. Using 6 differentgroups allows testing of at least 2 separate agents with at least 3different dosing regimens. In general, higher concentrations are usedfor shorter durations and lower concentrations for longer durations.Thus, for each group weekly time points with measurements of cornealthickness, endothelial cell parameters, keratocyte counts, andepithelial cell counts from the ConfoScan4 are available; and cornealthickness, IOP, corneal hysteresis, and corneal resistance factor fromthe ORA. This generates data in table form.

Most of the other aspects of this application involve simple comparisonsof group means which are analyzed statistically using simple t-tests. Inthis live rabbit study, the interest is in testing the null hypothesisof no treatment effect (the profiles of means [for y] are parallel inthe treatment group and the control group and also at the same level).The primary analysis method to be used is multivariate linear regressionthat separately models the means and the covariance structure. This isnecessary to estimate correct tests of hypotheses and confidenceintervals since the measurements are not independent (repeated measureswithin rabbits) of each other. The statistical program SAS “Proc mixed”are used to perform the analysis. An example program for this analysisis shown below:

[Proc mixed data=***; Class id trt week t; Model y=trt week trt*week/schisq; Repeated t/type=cs subject=id r;]

The main predictor is treatments (trt). The association of treatmenteffect and change in y is evaluated by the treatment*week interaction.Compound symmetry (CS) covariance structure is used if the covariancestructure matches the data obtained. If CS covariance structure does notmatch the data obtained, the best fitting model using maximum likelihoodtests for nested models and Akaike's information Criteria (AIC) fornon-nested models is selected. Covariates are not necessary since bothtreated and control data is nested in the same animal (i.e. left eyecontrol, right eye treated). Similar analytic strategies are used forother measurements b, c, d, . . . (i.e. thickness, IOP, etc.).

The nitro agent may be able to cross-link cornea in vitro and producebiomechanical stiffness yet be unable to produce the same effects in aliving eye. In this case, the agent may exhibit poor penetration throughthe corneal epithelium resulting in low or absent levels of agent in thestroma. In addition to the strategies outlined above, one alternative isto create a window or grid lines through the epithelium to allow passageof the compound into the stroma (this technique is used with UVRtherapy). In this case the cornea is saturated by continuous applicationwith the epithelium removed and thereby achieve high stromal tissueconcentrations. Since epithelial regeneration requires 3-4 days, theagent is applied daily or on an increased dosage schedule (i.e. threetimes per day or more) until the epithelium has reformed.

The agent may be able to cross-link cornea in vitro and producebiomechanical stiffness yet be unable to produce the same effects in aliving eye. The nitro agent can be photolyzed to increase effectiveness.As with riboflavin, nitrite (λ_(max)=350 nm) and other nitro compoundsundergo well-known photochemical reactions. Fischer, M. and Warneck, P.,“Photodecomposition of nitrite and undissociated nitrous acid in aqueoussolution,” J. Phys. Chem. 1996; 100:18749-18756. Iontophoresis and theuse of collagen shields are used to increase agent effectiveness. Thefirst methods require the use of an electrical charge device which isused to aid the passage of cross-linking compounds across the cornealepithelium and has been used to deliver antibiotics into the cornealstroma. Frucht-Pery, J., et al., “A. Iontophoresis-gentamicin deliveryinto the rabbit cornea, using a hydrogel delivery probe,” Exp. Eye Res.2004; 78:745-749. The second technique involves saturating a collagenshield with cross-linking agent and applying it to the cornea.Friedberg, M. L., et al., “Collagen shields, iontophoresis, and pumps,”Ophthalmology 1991; 98:725-732. Prolonged release from the shield couldthen occur over the course of several days.

Results

Efforts regarding the concept of nitrite induced collagen cross-linkinghave been directed at elucidating age-related damage mechanisms in humandisease. Indeed, age-related non-enzymatic collagen cross-linking is awell-known phenomenon occurring in numerous organ systems. Since humannitrite exposure can occur from various important sources such assmoking, inflammation, and diet, the finding that nitrite inducedcollagen cross-linking mimics aging changes has led to the hypothesisthat age-related tissue damage may result from lifetime nitriteexposures. The initial studies have documented increases in resistanceto enzymatic digestion (Paik, D. C., et al., “The nitrite/collagenreaction: non-enzymatic nitration as a model system for age-relateddamage,” Con. Tis. Res. 2001; 42(2):111-122), as well as increasedcollagen primary chain covalent cross-linking by SDS-PAGE (Paik, D. C.,et al., “Nitrite induced cross-linking alters remodeling and mechanicalproperties of collagenous engineered tissues,” Con. Tis. Res. 2006;47:163-176). It has been discovered that fibroblast-populated type Icollagen gels show an increase in tissue stiffening following nitritecross-linking. Although the conclusions drawn from these studiesimplicate nitrite cross-linking as an age-related deleterious process,the collagen cross-linking can have therapeutic applications.

Biomechanical Stiffening Changes Induced in Collagenous Tissues byCross-Linking by Nitrite

In order to study the biomechanical stiffening changes that can beinduced in collagenous tissues by cross-linking by nitrite, thefollowing study was undertaken. FIG. 3 provides a diagram and averagedeformation data showing the phases of the experiment for a uniaxiallyconstrained gel. Initially square fibroblast-populated collagen gelswere cast and allowed to polymerize for 2 hrs. Markers were painted onthe gel surface to allow tracking of deformation in the central regionduring the remainder of the experiment. For the next 72 hrs (remodelingphase), spontaneous contraction of the gel by the fibroblasts resultedin vertical compaction (dotted black line), while applied weightsprevented compaction in the horizontal direction (dotted gray line). At72 hrs, the gel was mechanically tested. First, the gel was loaded andunloaded several times (i.e. generally 5 cycles) until the responsestabilized (preconditioning phase). Then, the gel was loaded in a seriesof smaller steps and the response quantified (loading phase). Duringmechanical testing, deformations were much larger in the unconstrained,remodeled vertical direction (black line) than in the constrained,unremodeled horizontal direction (gray line).

Effects of Cross-Linking Porcine Cornea Using β-Nitro Alcohols

These are the results of studies dealing directly with cross-linking ofcorneoscleral tissue. FIG. 13 illustrates the gross effects ofcross-linking porcine cornea using β-nitro alcohols. These images weretaken from tissues cross-linked with nitrite and the β-nitro alcohols2-nitroethanol, 2-nitro-1-propanol, and 3-nitro-2-phenol, for 48 hrs.FIG. 14(A-E) shows treated porcine corneal strips. Fresh porcine cornealstrips and corneoscleral complexes were obtained within 6 hrs ofsacrifice and incubated at 37° C. in buffered solutions (pH 7.4)containing 20% Dextran (T500) and either 100 mM NaNO₂ or 100 mM of oneeach of the β-nitro alcohols. After 48 hrs, the following images wereobtained. FIG. 13 (A and C) A dramatic stiffness change is induced by2-nitroethanol and 3-nitro-2-pentanol when handled with a forceps. FIG.13 (B and D) An intermediate effect is seen with 2-nitro-1-propanol andNaNO₂. FIG. 13 (E) Control cornea show very little stiffness. FIG. 13illustrates the preserved transparency of the cornea treated withβ-nitro alcohols, note yellow tint in 2-nitroethanol treated cornea.This observation raised a concern that cross-linking using this compoundmay induce negative effects on an individuals' blue light perception. At100 mM concentration, a significant yellowing effect was observed using2-nitroethanol, a similar but much less dramatic effect was seen for2-nitro-1-propanol, and no yellowing effect was observable using3-nitro-2-pentanol. As such, the effects on corneal light transmissioninduced by cross-linking with β-nitro alcohols was evaluated using amethod adopted from Dillon et al 2004.

Following a 96 hrs incubation period at 10 mM, the corneo-scleralcomplexes were mounted between 2 pieces of quartz, previously determinedto have no ultraviolet absorption. This cross-linking regimen was chosenbecause the yellowing effect for 2-nitroethanol was profound at 100 mMmaking it impossible to obtain an absorption spectra at this level ofcross-linking. Further, it represents a target level of cross-linking asdetermined by thermal shrinkage temperature. The tissue cross-linkingeffects induced by these conditions are commensurate with those reportedfor UV/riboflavin cross-linking and as such represent a level ofcross-linking that would be predicted to have therapeutic value. Thesample was then mounted in a vertical, adjustable stage apparatusspecifically designed to accommodate a PC 1000 Fibre Optic Spectrometer(Ocean Optics, Inc.). A xenon lamp was used as a light source and wasconnected to the upper half of the stage. The lower half was connectedto a high sensitivity COD mounted on a card which is installed in a PCcomputer. The CCD array detector captures a full wavelength spectrum(i.e. 200-1000 nm).

The absorption spectrum for each sample was recorded and exported intoMicrocal Origin 6.1 for processing. Correction of the absorption spectrafor light scattering (i.e. Rayleigh and Tyndall) was then performed asdescribed by Dillon et al (2004). The scattering component wassubtracted from the observed absorption spectrum by first fitting thescattering (non-absorption) portion of the spectrum (e.g. 700-800 nm) tothe formula A=aλ^(b), where A is the absorbance, λ is the wavelength, bis the order of the relationship between absorbance and wavelength, anda is a constant. The background signal due to scattering for all otherwavelengths is then estimated using the coefficients determined from thefit. These values are then subtracted from the spectrum to generate thetrue absorption spectrum. After scatter correction, the transmissionspectrum for each absorption spectrum was calculated according to theBeer-Lambert law (T=10^(−A)×100%), where T is the transmission and A isthe absorbance at each wavelength. Finally, the effects on blue lighttransmission was determined by integrating the transmission spectra from400-500 nm. A percent decrease was then calculated with respect to thearea calculations using control samples.

A modest decrease in light transmission was noted for each compound.Decreased transmission was greatest for 2-nitroethanol, followed by2nprop, and 3n2pent. Integration of the 400-500 nm blue light regionrevealed decreases of 3.6%, 1.5%, and 1.0%, for 2-nitroethanol, 2nprop,and 3n2pent, respectively (FIG. 15). The tinting can be avoided by usingother β-nitro alcohols such as 2-nitro-1-propanol or 2-nitro-1-pentanol,both of which produce shifts in Ts without producing yellowing.

Shrinkage Temperatures of Porcine Cornea and Porcine and Human ScleraCross-Linked with β-Nitro Alcohols

Shrinkage temperature (T_(s)) of porcine cornea (FIG. 16) and porcineand human sclera (FIGS. 17 and 18) cross-linked with 2-nitroethanol.Fresh porcine corneal/sclera and human eye bank scleral strips (10×4 mm)were taken from the superior-inferior axis (cornea) and radially(sclera) and reacted with various concentrations of nitrite relatedagents (1-100 mM) for 24-96 hrs at 37° C. in 20% Dextran (T500), 0.2MNaH₂PO₄/Na₂HPO₄ (pH 7.4). Following reaction, thermal shrinkagetemperature analysis was performed (Table 4). Several compounds werestudied including NaNO₂, 2-nitroethanol, 2-nitro-1-propanol,3-nitro-2-pentanol, 2-nitrophenol, 2-nitroethane, 2-aminoethanol,isopentyl nitrite, DPTA/NO, DETA/NO, and urea, a nitrous acid trap.

Controls displayed an onset of T_(s) at 63° C. consistent with publisheddata from the UVR group. T_(s) onset were shifted 1-2° C. at 1 mM(serially applied over 6 days), 5° C. at 10 mM, and 8° C. at 100 mM2-nitroethanol in porcine corneas (FIG. 16). These levels ofcross-linking compare favorably with UVR reported T_(s) onset values (5°C. shift). T_(s) of porcine sclera are comparable to T_(s) determinedfor porcine cornea with a maximal increase of T_(s) of 8° C. at 100 mM2-nitroethanol. Two human donor globes were used in order to confirm theeffects of 2-nitroethanol in human tissue. As shown in FIG. 18, humansamples similarly treated showed a similar degree of shift in T_(s) ascompared to porcine tissue, demonstrating a correlation betweencross-linking porcine and human tissues. In addition, control humansclera showed similar T_(s) values to porcine although the total percentshrinkage was higher. These shifts in T_(s) indicate that nitrotechnology cross-links corneoscleral tissues.

Time related shrinkage temperature curves changes using 2-nitroethanolon porcine sclera are illustrated in FIG. 19. FIG. 19A. The graph showstime dependent shifts in T_(s) from porcine sclera cross-linked with 100mM 2-nitroethanol over the course of 96 hrs (time dependent effect).That is, a longer incubation results in a higher temperature ofshrinkage onset indicating increased cross-linking. A slightly differentkinetic experiment was also performed using 2-nitroethanol at aconcentration of 1 mM (FIG. 19B). In this case, the incubation washandled in a slightly different manner than the other experiments. Forthis study, the incubation solution containing the 1 mM 2-nitroethanolwas exchanged daily for the duration of reaction. This was performed inorder to replenish any of the reacted compound, maintaining a 1 mMconcentration over the duration of exposure. As shown in FIG. 19B, atime dependent shift in T_(s) was observed using a 1 mM concentration of2-nitroethanol with shifts in T₅₀ of 1.3, 3.2, 5.4° C., at 6, 10 and 14days, respectively. This concentration, by contrast, when reactedwithout exchange of the incubation fluid, did not induce a significantshift in T_(s) after 4 days (FIG. 17A). Further, in FIG. 20, using a 1mM 2-nitroethanol concentration, the incubation solution was changeddaily over the course of 10 days and compared to Ts changes producedthrough incubating with 10 mM 2-nitroethanol over 4 days and 100 mM2-nitroethanol over 3 days without changing the solution. This graphFURTHER indicates that comparable degrees of cross-linking can beachieved through modulation of reagent concentration and time ofexposure. In other words, a relatively lower concentration of2-nitroethanol (i.e. 1 mM) through serial applications (i.e. changingincubation fluid daily) can induce changes comparable to those observedusing higher concentrations (i.e. 10 mM) for shorter time periods (i.e.4 days). Simply using 1 mM 2-nitroethanol for 4 days without replacingthe incubation solution did not produce a shift in Ts in porcine corneaor sclera.

TABLE 4 Thermal shrinkage temperature changes induced in porcine (andhuman) scleral strips by reaction with β-nitro alcohols and relatedagents. Incubation solution includes 20% Dextran (T500) and 0.2MNaH₂PO₄/Na₂HPO₄ buffer pH 7.4 and 37° C. unless otherwise indicated.Incubation time was 96 hrs unless otherwise indicated. Temperatures areindicated in ° C. T₅₀ for each condition was compared to control values(Student's t-test) in order to determine p-values for statisticalsignificance. Each value is the average of a minimum of 3 independentdeterminations. reagent p-value concentration (for Compound/condition(mM) T_(i) ^(*) T₅₀ ^(†) T₅₀Δ^(‡) T₅₀) 2-nitroethanol 100 70.7 73.7 8.40.000 10 67.2 69.8 4.5 0.000 1 62.9 65.7 0.4 NS 2-nitro-l-propanol 10069.5 72.7 7.4 0.000 10 64.4 67.5 2.2 0.013 1 63.0 66.0 0.7 NS3-nitro-2-pentanol 100 68.3 70.7 5.4 0.001 10 65.3 67.7 2.4 0.006 1 62.566.2 0.9 NS 2-nitrophenol 100 62.9 66.0 0.7 NS 10 62.3 65.6 0.3 NS 162.2 65.2 −0.1 NS 2-nitroethane 100 62.5 65.3 0 NS 10 62.5 65.4 0.1 NS2-aminoethanol 100 64.3 67.9 2.6 0.011 (ethanolamine) Control porcinesclera 62.9 65.3 (4° C.) Control human sclera (4° C.) 61.2 66.0 Humansclera 2-nitro 100 71.7 73.5 7.5 0.000 ethanol 10 64.4 68.2 2.2 0.026 160.8 66.3 0.3 NS ^(*)T_(i) = temperature at 1% absolute shrinkage,^(†)T₅₀ = temperature at 50% of maximal shrinkage (or maximal rate ofshrinkage change), ^(‡)T₅₀Δ = change in 50% shrinkage temperaturecompared to control. All values were compared to porcine controls exceptfor studies using human sclera and 2-nitroethanol.

Additional β-nitro alcohols related to 2-nitroethanol were also testedfor scleral cross-linking efficacy. In addition to 2-nitroethanol, therewas 2-nitro-1-propanol, 3-nitro-2-pentanol, and 2-nitrophenol. Of these,the short chain aliphatic compounds were shown to be effectivecross-linking agents. 2-nitroethanol showed the greatest efficacy,shifting the T₅₀ by 8.5° C. at 100 mM concentration in porcine scleraand 7.5° C. in human sclera at 96 hrs of incubation (FIG. 18). Thiseffect was not blocked by addition of equimolar urea (Table 5).2-nitro-1-propanol (FIG. 17B) and 3-nitro-2-pentanol (FIG. 17C) showedcross-linking efficacy as well, with a T₅₀ shift for 2-nitro-1-propanoland 3-nitro-2-pentanol of 7.4° C. and 5.4° C., respectively. All threeof these short chain aliphatic compounds showed a concentrationdependent effect. Of note, 2-nitroethanol appears to have the greatestpropensity to induce tissue yellowing. This appears to be aconcentration dependent effect with the order of greatest yellowing indecreasing order: 2-nitroethanol, 2-nitro-1-propanol, and3-nitro-2-pentanol. Very little yellowing was noted for any of thesamples reacted at 10 mM.

No shift in T_(s) is noted at 1 mM concentrations of either compound.This is similar to 2-nitroethanol in which shifts in T_(s) at 1 mM couldonly be produced through serial application (i.e. daily changing ofincubation solution) over the course of several days. The higherconcentrations produce shifts in T_(s) somewhat less than those foundusing 2-nitroethanol.

Several compounds were found to be ineffective as cross-linking agents.2-nitrophenol, an aromatic β-nitro alcohol, produced very little shiftin T_(s) (FIG. 17D). 2-aminoethanol, the β-amino alcohol correspondingto 2-nitroethanol, also produced only a marginal shift in T_(s) (Table1). 2-nitroethane, the nitroalkane corresponding to 2-nitroethanol, didnot shift the T_(s) to any extent (FIG. 21) (Table 4), indicating thatthe presence of the alcohol group was an important moiety in thesereactions.

The results from cross-linking using glutaraldehyde and formaldehydeallow for a comparison to be made with this prototype chemicalcross-linking agent. At moderate levels of T₅₀ shift (i.e. ˜4° C.),glutaraldehyde was approximately 10× more potent as a cross-linkingagent that 2-nitroethanol, producing a 3.6° C. shift at 1 mMconcentration versus 4.5° C. shift at 10 mM concentration for2-nitroethanol (see Table 5 and Table 4 respectively).

(Table 5) Previous studies have indicated that although the reactionwith free nitrite at neutral pH can result in collagen cross-linking,the effects are slow, taking on the order of weeks. Furthermore, thesecross-linking effects were shown to be facilitated by reacting underacidic conditions Paik D C, “Nitrite induced cross-linking altersremodeling and mechanical properties of collagenous engineered tissues”.Connect Tis Res. 47:163-76. In the present study, the effects of pH onthe nitrite induced cross-linking were studied by reacting at threedifferent pH values, 3, 5, and 7.4. At 96 hours, no effect was seen atpH 7.4. However, at pH 3 and 5, approximately equal shifts in T_(s) wereobserved. Acid alone in the absence of NaNO₂ destabilized the tissuesand resulted in a downward shift in T_(s) (data not shown). The factthat acidification of NaNO₂ would increase cross-linking suggested thatnitrosation could be involved Williams D L H. Nitrosation Reactions andthe Chemistry of Nitric Oxide. Amsterdam, The Netherlands: Elsevier B.V.; 2004:1-43.

However, additional experiments did not support this claim. Equimolarurea a well-known nitrous acid trapping agent Fitzpatrick J, et. al,“Comparison of the reactivity of nine nitrous acid scavengers”. J ChemSoc Perkin Trans II. 1984:927-32, was unable to block the cross-linkingeffect by NaNO₂ at pH 3. In addition, studies using the nitrite esterisopentyl nitrite at neutral pH, an effective nitrosating agent, did notresult in a shift in T_(s) (FIG. 22). In these experiments, becauseisopentyl nitrite has poor solubility in water, a small facilitatingamount of an organic solvent was added to increase solubility. Bothethanol and DMSO were used for this purpose, but neither conditionresulted in changes in T. Finally, recent studies by Kong et al (2006)have shown that NO donors of the diazeniumdiolate class can result innitrosation of peptide amide moieties resulting in deamidation, Kong L,Saavedra J E, Buzard G S, et al. “Deamidation of peptides in aerobicnitric oxide solution by a nitrosative pathway”. Nitric Oxide. 2006;14(2):144-51. Thus, we also tested these compounds for cross-linkingefficacy. At neutral pH, DPTA/NO with a t/2=3 hrs was reacted for 24 hrsand DETA/NO with a t/2=30 hrs was reacted for 96 hrs. Neither compoundwas able to shift T_(s) parameters (FIG. 23 A and B).

TABLE 5 Thermal shrinkage temperature changes induced in porcine scleraby reaction with acidified NaNO₂, nitrosatingagents, aldehydes, andother agents. Incubation solution includes 20% Dextran (T500) and 0.2MNaH₂PO₄/Na₂HPO₄ buffer pH 7.4 and 37° C. unless otherwise indicated.Incubation time was 96 hrs unless otherwise indicated. Temperatures areindicated in ° C. T₅₀ for each condition was compared to control values(Student's t-test) in order to determine p-values for statisticalsignificance. Each value is the average of a minimum of 3 independentdeterminations. reagent p-value Compound/condition concentration T50 T50Δ (for T50) NaNO2 pH 3 100 mM 69.3 4.0 <.01 NaNO2 pH 5 100 mM 67.3 2.0<.01 NaNO2 pH 7.4 100 mM 65.7 0.4 NS NaNO2 100 mM pH 3 100 mM urea 65.60.3 NS  10 mM urea 69.7 4.4 <.01  1 mM urea 70.1 4.8 <.01 Isopentylnitrite (1% DMSO) 100 mM 65.4 0.1 NS DPTA/NONO (24 hrs reaction) 100 mM66.2 0.9 NS  10 mM 66.4 1.1 NS DETA/NONO (96 hrs reaction) 100 mM 65.50.2 NS Glutaraldehyde  1 mM 68.9 3.6 <.01 Formaldehyde  1 mM 67.7 2.4<.01 Ethanol 100 mM 65.2 −0.1 MS DMSO  1% 65.0 −0.3 NS Control porcinesclera (4oC) 65.3 Control human sclera (4oC) 66.0 ^(*)T_(i) =temperature at 1% absolute shrinkage, ^(†)T50 = temperature at 50% ofmaximal shrinkage (or maximal rate of shrinkage change), ^(‡)T50 Δ =change in 50% shrinkage temperature compared to control. All values werecompared to porcine controls.

A cytotoxicity study was undertaken to obtain an impression of the levelof cellular tolerance to these compounds. For pilot experiments ARPE-19cells were used. This is an immortal human retinal pigment epithelialcell line commonly used for experiments related to macular degeneration.In these pilot studies, the ARPE-19 cells serve as a surrogate forcytotoxicity experiments. Primary cultures of bovine corneal endothelialcells were also used in the cytotoxicity studies described. Bovinecorneal cells have more direct relevance for evaluating potentialcytotoxic effects during cross-linking of living eye tissue.

ARPE-19 cells were grown to 80% confluence in 96 well tissue cultureplates. The cells were then exposed to varying concentrations of NaNO₂and 2-nitroethanol in their culture medium and evaluated for cell deathusing trypan blue (TB) staining (0.4% for 5 min) after 24 hrs. FIG. 24shows the treated cells. FIG. 24(A) shows the morphology of controlcells which did not stain positive with TB. A threshold of toxicity wasfound for cells exposed to 10 mm 2-nitroethanol. FIG. 24(D) shows thatall of the cells took up TB in the 10 mM 2-nitroethanol group. FIG.24(B) shows that no TB was taken up by cells at 1 mM 2-nitroethanol.FIG. 24(C) shows that even high levels of NaNO₂ (i.e. 100 mM) weretolerated by cells. These experiments indicate that there is a cytotoxicthreshold for 2-nitroethanol that is between 1-10 mM. It appears thateven millimolar levels of 2-nitroethanol can be tolerated by livingcells.

Bovine corneal cells have are more directly relevant for the evaluationof potential cytotoxic effects during cross-linking of living eyetissue. As such, primary bovine corneal cells were used in cytotoxicitystudies of β-nitro alcohols.

After growing in 96-well plates and with cells at 80-90% confluence,compounds of interest were added into the culture medium at a range ofconcentrations (0.001-1%). Following a 48 hrs incubation period, a cellsuspension was obtained by trypsinization and dead cells were stainedusing 0.4% trypan blue for 5 min. The live/dead cells were then countedusing a hemocytometer and the dead cell percent calculated. Three wellswere used for each condition. A similar protocol was performed toevaluate the degree of apoptosis and necrosis, utilizing annexin V andpropidium iodide staining according to the manufacturer's protocol(Molecular Probes, Inc.).

The toxicity levels vary between different β-nitro alcohols, wherein thehighest tolerated level of 2-nitroethanol and 3-nitro-2-pentanol is 2 mMand the highest tolerated level of 2-nitro-1-propanol is 0.75 mM (FIG.25, Tables 1 and 2). Comparatively, at 1 mM concentration, T_(s) forporcine cornea was shifted 1-2° C. when β-nitro alcohols were seriallyapplied over 6 days (FIG. 16) and T_(s) for porcine sclera was shifted2-3° C. when serially applied over 10 days (FIG. 19). The cytotoxicthreshold of the β-nitro alcohols compares favorably with other knownophthalmic agents (Table 3).

For example, genipin, a natural iridoid cross-linking agent derived fromthe gardenia plant, has been reported to exhibit low cytotoxicity. Incytotoxicity studies using human fibroblasts, tolerable levels ofgenipin (0.44 mM or 0.01%) were shown to be more than 100× greater thanglutaraldehyde and more than 20× greater than epoxy. Sung, H. W., etal., “Feasibility study of a natural crosslinking reagent for biologicaltissue fixation,” J. Biomed. Mater. Res. 1998; 42:560-567. By comparisonwith the genipin studies, these cytotoxicity studies use the sameexposure time (i.e. 24-48 hrs) with retinal pigment epithelial cells(ARPE-19) and bovine corneal endothelial cells. As shown above and inTable 3, the tolerable level of 2-nitroethanol was comparable in ARPE-19to those reported for genipin (1 mM or 0.0091% for 2-nitroethanol vs.0.44 mM or 0.01% for genipin). In bovine corneal endothelial cells thetolerable level of 2-nitroethanol was greater than those reported forgenipin (3 mM or 0.0273% for 2-nitroethanol. Based on these studies, itis believed that serial application using lower doses can producecross-linking effects similar to those induced at higher concentrationsfor shorter incubation periods. This therapy may produce cornealcross-linking over a relatively long period (2-8 weeks) using a dose ofdrops that would produce a subtoxic stromal tissue level. For freenitrite the tolerable levels were at least 100× higher (100 mM) thangenipin, indicating that these compounds are relatively well toleratedby cells. β-nitro alcohols have very favorable mutagenicity (Conaway, C.C., et al., 1991) and animal toxicity profiles (Jung, Y. S., et al.,2004). As such, they have been proposed for use in animal feeds in orderto control food borne pathogens in ruminants and chickens where theyexhibit bacteriostatic activity (Horrocks, S. M., et al., 2007).Nitrite, as a sodium salt, can be found in the typical American andEuropean diet, where it is routinely used as a cured meat preservative(Walker, R., 1990).

Penetration through the corneal epithelium is an important considerationregarding the utility of any potential topical therapy. In order toobtain an initial impression regarding the ability of nitrite andrelated compounds to pass through the corneal epithelium the followingexperiment was undertaken, the results of which are illustrated in FIG.5.

Adult porcine eyes were obtained within 12 hrs of sacrifice andsubmerged in solutions of 0, 10, 100 mM NaNO₂ and 100 mM NaCl bufferedwith 50 mM NaH₂PO₄/Na₂HPO₄ (pH 7.4) following bubbling with 100% Argonin order to limit the amount of potential nitrite auto-oxidation causedby oxygen. Penicillin/Streptomycin was added (10 ul/ml) to preventbacterial overgrowth. After 24 hrs of incubation at 4° C., the aqueoushumor was sampled for nitrite concentration using a modification of theGreiss colorimetric assay. No nitrite was detected in either the buffercontrol or the 100 mM NaCl control. Nitrite concentration in the aqueoushumor was 35.5% of the incubation fluid in the 10 mM NaNO₂ sample and62.8% in the 100 mM NaNO₂ sample. These results suggest that there maybe a concentration dependent penetration of nitrite through the cornea.

Mechanistic and Catalytic Studies of β-Nitro Alcohol (βNA) Cross-Linkingwith Amine Functionalized Polymers

The present studies were undertaken to investigate the underlyingreactions and mechanisms involved in β-nitro alcohol (βNA) cross-linkingof collagenous tissues and to characterize the mechanisms by which βNAsreact with amine functionalized polymers. The amine functionalizedpolymer poly(allylamine) (PAA) was used as a surrogate model substrateto investigate the mechanism by which βNAs crosslink collagen incollagenous tissues. The specific cross-linking sites for both PAA andcollagen are primary amine groups. In the case of collagen, this primaryamine is the epsilon amine found in both lysine and 5-hydroxylysineresidues.

Upon the reaction of a solution of PAA with βNAs a hydrogel is produced.The resulting hydrogel may be used as a mechanistic signature todetermine the extent of cross-linking of the PAA. Similarly, thecorresponding increase in the mechanical strength of the hydrogel thatis observed upon cross-linking of the PAA substrate may be used as ameans of determining the extent of cross-linking of the PAA.

In the present studies, the reaction and the involved products orspecies were further examined by ¹H-NMR and FTIR analysis to gainfurther insight regarding the underlying reaction and the ability ofβNAs to crosslink collagenous tissues, as well as to determine theimpact that select catalysts have on the rate and efficiency ofβNA-mediated cross-linking of collagenous tissues.

Materials and Method:

Poly(allylamine) hydrochloride (PAA) (Aldrich, average MW ˜15,000);

Sodium phosphate monobasic (NaH₂PO₄, Aldrich, 99%);

Sodium phosphate dibasic (Na₂HPO₄, Aldrich, 99%);

2-nitro-ethanol (Aldrich, 97%);

2-methyl-2-nitro-1,3-propanediol (Alfa, 97%);

tris(hydroxymehyl)nitro methane (Aldrich, 98%);

2-bromo-2-nitro-1,3-propanediol (Aldrich, 98%);

Deoxyribonucleic acid sodium salt, from salmon testes (Aldrich);

Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) (Aldrich); and

Paraformaldehyde (Aldrich, 95%).

All of the materials were used as received. A buffer solution (pH 7.4)was prepared by mixing 81% of 0.2M Na₂HPO₄ and 19% of 0.2M NaH₂PO₄aqueous solution. The structures of those βNAs investigated in thepresent studies are identified in Table 6 below, as are the aminefunctionalized polymer poly(allylamine) (PAA) and paraformaldehyde(PFA).

TABLE 6 The chemical structures, names and abbreviations ofrepresentative β-Nitro alcohols, paraformaldehyde and poly(allylamine).Structure Chemical Name Abbreviation

2-hydroxymethyl-2- nitro -1,3-propanediol (nitro-triol) HNPD

2-methyl-2-nitro-1,3- propanediol (nitro-diol) MNPD

2-nitroethanol 2NE

2-nitro-1-propanol 2NPROP

2-Bromo-2-nitro-1,3- propanediol BRONOPOL

paraformaldehyde PFA

Poly(allylamine) PAA

Equipment:

Nicolet Nexus 870 FTIR with a Thermo Multibounce HATR accessory with aZnSe crystal;

WTW Measurement System, Chekmite pH-20 sensor; and

Bruker 400 MHz NMR spectrometer.

To determine the extent of substrate (PAA) cross-linking, the rate offormation of the hydrogel was qualitatively measured by the time takenfor a liquid polymer substrate (PAA) sample undergoing gel formation tomaintain its shape against gravity. As illustrated in FIG. 28, prior tobeing washed and dried, a gel sample in a vial containing the resultanthydrogel can initially maintain its shape for hours after inversion ofthe vial.

In the present study PAA (110 mg) and the βNAs evaluated (Table 6) weredissolved in 0.3 mL of pH 7.4 phosphate buffer solution and the mixturewas heated to 37° C. Gel precipitates were formed after several hours.The reaction was maintained until a firm gel was formed. Unreacted βNAand PAA were then removed by washing with a solution of ethanol andwater. Various concentrations of ethanol in water (100%, 50%, 30%, and0%) were used for the washings.

The washed gels were dried under vacuum overnight at room temperatureand the weight of water absorption and the degree of swelling wererecorded. Swelling experiments were conducted in H₂O as shown in FIG.28. Approximately 40 mg of the dried gel was immersed into 5 mL water atroom temperature for 1 hour. The weights of the swollen samples werethen measured after the excess surface water was removed by filterpaper. The degree of swelling was calculated using the swell ratio W/W₀,where W and W₀ are the weights of the swollen and dried gels,respectively, and are shown in Table 7 below.

The washed gels were then characterized by FTIR. FIGS. 29 and 30 depictFTIR spectra of the hydrogels that resulted from the cross-linkingreactions. The FTIR spectra of the hydrogels were taken immediatelyafter reaction (dashed lines) and the hydrogels were washed withwater/ethanol, dried and the FTIR spectra were taken again (solidlines).

As a control experiment, PAA was reacted with parafomaldehyde (PFA),which releases formaldehyde in solution. The IR peak at 1632 cm⁻¹ of theresulting hydrogel before washing in FIG. 29 (dashed line) was assignedto the carbonyl group (—C═O) of formaldehyde. As illustrated in FIG. 29,the peak disappeared after washing with the water/ethanol solution anddrying (solid line).

Cross-linking experiments of PAA using the βNA2-hydroxymethyl-2-nitro-1,3-propanediol (nitro-triol or HNPD) were alsoperformed under the same conditions. The IR peaks at 1540 cm⁻¹ and 1070cm⁻¹ depicted in FIG. 30 (dashed line, before washing) were assigned toNO₂ symmetric stretching and C—NO₂ bending, respectively. There was alsoa peak at 1632 cm⁻¹, which was assigned to a C═O stretch. These peaksdisappeared after washing and drying of the hydrogel. The FTIR spectraof the hydrogel before washing and drying (dashed line) has the samecharacteristic peak corresponding to the C═O group that was assigned toformaldehyde, thereby indicating that a carbonyl group was produced inboth of the cross-linking reactions.

The foregoing results are consistent with the release of formaldehydeduring the PAA cross-linking reaction using both paraformaldehyde andthe βNA nitro-triol as cross-linking reagents. The similar IR spectra ofdried hydrogel (solid line) observed for both reactions indicate that a′similar cross-linking product was formed. Furthermore the absence of anIR peak for the nitro group after drying of the hydrogel demonstratesthat the cross-linking occurred between the PAA substrate and theformaldehyde which was released from the PFA or from the nitro-triol andthat the nitroalkyl did not participate in the cross-linking of the PAAsubstrate.

To further confirm the formation of formaldehyde during the PAAsubstrate cross-linking reaction, ¹H-NMR spectra were taken to provide adirect measurement of formaldehyde, which exhibits a characteristicchemical shift around 8.5 ppm. FIG. 31 depicts the ¹H-NMR spectrum ofnitro-triol after 20 hours at 37° C. and pH 12.7 (top). The chemicalshift observed at 8.48 and depicted in FIG. 31 indicates the generationof formaldehyde from the βNA nitro-triol. Similarly, FIG. 32 provides acomparison of ¹H-NMR spectra of formaldehyde produced by thebase-catalyzed decomposition of the βNA nitro-ethanol. The observedconcentrations of formaldehyde released from the higher order βNAnitro-triol was higher than that observed from the βNA nitro-ethanol.This observation is due to the fact that the βNA nitro-triol possessesthree potential equivalents of formaldehyde, relative to the oneequivalent of formaldehyde for the βNA nitro-ethanol.

The amount of formaldehyde did not change with increasing time uponcomparing the integration of chemical shift of formaldehyde and βNA.This observation suggests that formaldehyde release occurs through abase-catalyzed and thermally driven reverse Henry reaction and continuesuntil the reaction reaches equilibrium. As illustrated in FIG. 26, thereaction which results in the formation of formaldehyde from the βNAstarting materials is reversible.

As shown in FIG. 26, in the presence of base (B) the βNA undergoesde-protonation at the hydroxyl group (—OH), followed by the subsequentdecomposition of the βNA to yield formaldehyde by a base-catalyzedreverse Henry reaction. The liberated formaldehyde represents the activecross-linking agent capable of participating in the cross-linking ofcollagenous tissues.

The proposed mechanism by which βNAs crosslink PAA is depicted in FIG.27. As illustrated in FIG. 27, an amino group of the PAA side chain withthe lone electron pair attacks the electrophilic carbonyl carbon offormaldehyde from the βNA to form a Schiff base, which can then reactwith another PAA and formaldehyde to crosslink the PAA.

Next, the degree of gel formation and cross-linking induced upon thereaction of different βNAs with PAA was evaluated. The reactionconditions were optimized by using a mass ratio of βNA (e.g.,nitro-diol) to polymer (PAA) of 1:1. For example, according to theequivalent number of hydroxyl groups of βNAs compared with nitro-diol,60 mg of nitro-triol and 220 mg of mono-nitro-alcohol were used,respectively with 110 mg PAA substrate. The rate of gelation (i.e., theextent of cross-linking) observed in the PAA substrate solutiondecreased with the number of hydroxyl groups in the βNA evaluated in thefollowing order: triol, diol, and monol.

Additionally, the substituents of the βNAs evaluated were also capableof influencing the rate at which the substrate (PAA) solution underwentgelation. In particular, it was found that those substituents havingelectron-donating groups decreased the rate of gelation of the substrate(PAA) solution. For example, it took two weeks for themethyl-substituted nitro-propanol to form gels, relative to a gelationtime of 48 hours for the unsubstituted nitro-ethanol. The nitro-diol wasexpected to have a faster time of gelation relative to the mono-alcoholnitroethanol. However, the methyl-substituted nitro-diol significantlyslowed down the rate of gel formation, having a time of 66 hourscompared to the gel formation time of 48 hours that was observed fornitroethanol. This observation was attributed to the fact that themethyl group was an electron-donating substituent, which made theintermediate unstable and decreased the reaction rate. Conversely, asubstituent with electron-withdrawing substituents increased the rate ofgelation and the equilibrium favored the cross-linking reaction and theproduction of the hydrogel due to the stabilized intermediate as shownin Reaction 1 above.

As depicted in Table 7 below, 2-bromo-2-nitro-1,3-propanediol(BRONOPOL), which has a bromine substituent, formed gels in 24 hours,which was faster relative to the 66 hours observed for themethyl-substituted nitro-diol.

TABLE 7 The gelation time and the degree of swelling of hydrogelsprepared with nitro-alcohols and poly(allylamine). The amount of PAA waskept to 110 mg for all experiments. The experiment conditions were 37°C. and pH 7.4. The error of the measured time was 20 minutes. ReactantsAmount Gelation time Degree at (with PAA) (mg) (h) swelling

paraformaldehyde 19 24 25 ± 4

nitro-triol 60 29 28 ± 5

nitro-diol 110 66 22 ± 4

nitro-ethanol 220 48 21 ± 3

nitro-propanol 220 2 weeks (partial gel) 20 ± 3

Bronopol 110 42 15 ± 2

The ability of the PAA substrate to form gels upon exposure toparaformaldehyde (PFA) was also evaluated. As shown in Table 7, the gelformation time observed for PFA was 24 hours, which was faster than thatobserved for the nitro-triol. This observation further indicates thatformaldehyde is the direct cross-linking agent that is released fromβNAs.

Synthetic and biological cross-linked materials may swell when exposedto low molecular weight solvents. Accordingly, the degree of swelling ofthe hydrogels was also used as a measure of the degree of cross-linkingof the hydrogels. The degree of swelling at equilibrium depends onseveral factors such as, for example, temperature, length of the networkchains, number of the cross-links, size of the solvent molecules, andthe strength of thermodynamic interaction between the polymer chains andsolvent molecules.

The present hydrogel swelling studies were performed in deionized H₂O atroom temperature. As shown in FIG. 28 and Table 7, no significantdifference in the degree of swelling for the hydrogels produced by βNAsand PAA was observed. These results indicate that vibration of theconstituent —C—N bond of the polymer network cannot make much differencein length. This further supports that the observed cross-linking wasgenerated from the reaction of formaldehyde and PAA, as evidenced by theprevious IR results.

As shown in FIG. 27, the cross-linking of βNAs with PAA can beessentially understood as the reaction of formaldehyde with the PAA. Thesmall difference in the degree of swelling observed for BRONOPOLcross-linking may be a result of more cross-links relative to the otherβNAs. For polymers with the same structure and solvent, a larger numberof cross-links cause a smaller variation of chain length between thenetwork junctions. Therefore, a smaller extent of these chains yields asmaller degree of swelling as volume increases.

Effect of Catalysts on the Degree of Cross-Linking and Gel Formation

The base-catalyzed and reversible Henry reaction, which consists of acondensation of aliphatic nitro-compounds with aldehydes or ketones,represents a method for the synthesis of aliphatic nitro-alcohols (e.g.,βNAs). Conversely, the reverse Henry reaction of βNAs releasesformaldehyde that can act as a cross-linking agent and induce theformation of a hydrogel in PAA substrate solutions. Although strongbases like NaOH or KOH catalyze the reverse Henry reaction, such basesmay not be suitable for in vivo applications (e.g., topical orophthalmic administration), and accordingly in certain embodiments mildbases may be more suitable catalysts.

The present study was conducted to investigate the ability of catalyststo drive the reverse Henry reaction and to cause the gelation of PAAsolutions upon exposure to βNAs. NaHCO₃, DNA, and hydroxynitrile lyasewere each evaluated for their ability to catalyze the cross-linkingreaction of a PAA solution. The PAA cross-linking was performed inphosphate buffer solution (pH 7.4) at physiological temperature of 37°C.

As illustrated in Table 8 below, upon increasing the amount of NaHCO₃aqueous solution (0.5M, pH 9.0), the gel formation times for the PAAsolution significantly decreased relative to the reaction times observedin the absence of NaHCO₃. As would be expected, the pH of the phosphatebuffer solution increased upon the addition of NaHCO₃ aqueous solution.Therefore, the foregoing results illustrate that an increase in pH(i.e., basic conditions) is capable of catalyzing the cross-linking ofPAA and increasing the rate at which the reaction solution undergoesgelation.

TABLE 8 Effect of NaHCO3, salmon testes DNA, and hydroxynitrile lyase ongel formation of nitro-alcohols crosslinking reaction. For experimentswithout catalyst, 0.3 ml pH 7.4 phosphate buffer solution was used. ThepH of the reaction mixture was 8.2 when 0.05 ml of 0.5M NaHCO3 wasadded, and the pH was 8.0 when 0.025 ml of 0.5M NaHCO3 was added.Reaction Reaction time Reaction time time Reaction time with withwithout With salmon testes hydroxynitrile Nitro-alcohols catalyst 0.5MNaHCO₃ DNA lyase

nitro-triol 29 h  0.05 ml 0.025 ml   14 h 17.5 h 3.4 mg 26 h 0.1 ml 40 h

nitro-diol 66 h  0.05 ml 0.025 ml   29 h   36 h 3.1 mg 46 h 0.1 ml 96 h

bronopol 42 h  0.05 ml 0.025 ml   6 h   7 h 3.6 mg 18 h 0.1 ml 72 h

The ability of DNA obtained from natural sources to catalyze the reverseHenry reaction was also evaluated. Salmon testes DNA was selected as thecatalyst for the cross-linking reaction of the PAA solution, and thecorresponding gelation times are shown in Table 8. The rates of gelformation for each of three βNAs evaluated were observed to increaserelative to the reactions conducted in the absence of catalyst. Inparticular, the gelation time observed for2-bromo-2-nitro-1,3-propanediol (BRONOPOL) decreased significantlyrelative to the reaction conducted in the absence of catalyst. Relativeto the catalytic effects observed with NaHCO₃, the catalytic ability ofsalmon testes DNA appeared to be less. The foregoing results indicatethat salmon testes DNA also represent a suitable catalyst capable ofpromoting the cross-linking of PAA or collagenous tissues using βNAs.

The use of enzymes as catalysts of the reverse Henry reaction was alsoevaluated, and due to their mild reaction conditions may also representa suitable class of catalysts useful for promoting the cross-linking ofcollagenous tissues using βNAs. As shown in Table 8, 0.1 mLhydroxynitrile lyase containing an α/β-hydrolase fold from thenon-cyanogenic plant Arabidopsis thaliana (AtHNL) was added into the 0.3mL pH 7.4 buffer reaction mixtures of the βNAs and PAA, respectively.The observed reaction rates increased in the presence of hydroxynitrilelyase relative to those rates observed in the absence of catalyst. Theseresults indicate that hydroxynitrile lyases catalyze the formation ofthe βNA, rather than catalyzing the formation formaldehyde and thesubsequent cross-linking of the PAA solution. It is likely thatα/β-hydrolase plays a dominant role in observed the catalytical effect.

Discussion

Recent studies indicate that collagen cross-linking through reactionswith nitrite and related agents can induce corneal changes commensuratewith UVR therapy. In addition, published toxicity/mutagenicity studiessuggest a good safety profile for these agents. Thus, nitro technologycould be used as an “eye drop alternative” to UVR treatment. There areseveral advantages to this alternative. First, if no UVA irradiation isnecessary, this could lessen the degree of cytotoxicity. This couldallow doctors the opportunity to offer cross-linking treatment to allindividuals, even those with thin corneas<400 μm. Second, patients wouldexperience less discomfort since epithelial debridement would not benecessary. Third, since the compound would be self-administered,patients would benefit from the ease of application and reduced costs.Fourth, a more complete cross-linking could be possible, since watersoluble nitro compounds can diffuse easily through the corneal stroma.The UVR method cross-links only the anterior 200 μm of cornea whichcorrelates with the depth of penetration of UVA irradiation into theriboflavin soaked cornea (Kohlhaas, M., et al., 2006). Fifth, multiplere-treatments would be possible with topical therapy which may not bethe case for UVR treatment. Sixth, a dose modulation could supply aneffect of controlled magnitude rather that the single effect currentlyproduced with the UVR procedure.

Recent studies indicate that collagen cross-linking through reactionswith nitrite and related nitro agents can induce corneal collagenchanges commensurate with the UVA/riboflavin (UVR) therapy.Specifically, changes in thermal shrinkage temperature (a generalmeasure of collagen cross-linking) induced in pig cornea/sclera andhuman sclera, are comparable to that reported for UVR treatment. Inaddition, preliminary cell toxicity studies and literature suggest areasonable safety profile for these agents. Related nitro agents havevery favorable mutagenicity (Conaway, C. C., et al., “Evaluation ofsecondary nitroalkanes, nitrocarbinols, and other aliphatic nitrocompounds in the Ames Salmonella assay,” Mut. Res. 1991; 261(3):197-207)and animal toxicity profiles (Jung, Y. S., et al., “Experimental use of2-nitro-1-propanol for reduction of Salmonella Typhimurium in the cecaof broiler chicks,” J. Food Prot. 2004; 67:1945-1947). As such they havebeen proposed for use in animal feeds in order to control food bornepathogens in ruminants and chickens where they exhibit bacteriostaticactivity (Horrocks, S. M., et al., “Effects of short-chainnitrocompounds against Campylobacter jejuni and Campylobacter coli invitro,” J. Food Sci. 2007; 72(2):M50-M55). Finally, nitrite, as a sodiumsalt, can be found in the typical American and European diet, where itis routinely used as a cured meat preservative (Walker, R., “Nitrates,nitrites and N-nitrosocompounds: a review of the occurrence in food anddiet and the toxicological implications,” Food Additives andContaminants 1990; 7(6):717-768).

Thus, nitro technology may find clinical utility as a topical cornealstiffening agent and could have a significant impact not only on thetreatment of keratoconus (which affects younger individuals) but also onpost-PRK and post-LASIK keratectasias, which are devastatingcomplications of keratorefractive surgery. These latter mentionedkeratectasias are now emerging as a significant long-term complication(5-10 years) of LASIK and PRK surgery of unknown epidemiologicproportions (Binder, et al., 2005). They are also the basis of many oftoday's PRK- and LASIK-related medical malpractice litigations inophthalmology and optometry.

The earliest work from Wollensak, Spoerl, and Seiler was reported in1998. The initial studies were aimed at identifying methods useful forcorneal collagen cross-linking and included riboflavin with lightexposure, glutaraldehyde, formaldehyde, and other aldehyde sugars.Spoerl, E., et al., “Induction of cross-links in corneal tissue,” Exp.Eye Res. 1998; 66:97-103; Spoerl, E. and Seiler, T., “Techniques forstiffening the cornea,” J. Refract. Surg. 1999; 15:711-713. Thesestudies were followed by reports which determined the cytotoxic dose ofthe treatment on corneal endothelial cells and keratocytes using invitro cell culture (Wollensak, G., et al., “Corneal endothelialcytotoxicity of riboflavin/UVA treatment in vitro,” Ophthalmic. Res.2003; 35:324-328; Wollensak, G., et al., “Keratocyte cytotoxicity ofriboflavin/UVA-treatment in vitro,” Eye 2004; 18:718-722) and the rabbitas a test animal (Wollensak, G., et al., “Endothelial cell damage afterriboflavin-ultraviolet-A treatment in the rabbit,” J. Cataract Refract.Surg. 2003; 29:1786-1790; Wollensak, G., et al., “Collagen fiberdiameter in the rabbit cornea after collagen crosslinking byriboflavin/UVA,” Cornea 2004; 23:503-507). Simultaneously, studies wereperformed which examined biochemical properties of cross-linked cornealtissue. Basic studies examining thermal denaturation temperature(Spoerl, E., et al., “Thermomechanical behavior of collagen-cross-linkedporcine cornea,” Ophthalmologica 2004; 218:136-140) and resistance toenzymatic digestion (Spoerl, E., et al., “Increased resistance ofcrosslinked cornea against enzymatic digestion,” Cur. Eye Res. 2004;29(1):35-40) indicated that the combination of UVA with riboflavin as aphotosensitizer was effective in cross-linking corneal collagenlamellae. These studies were performed in conjunction with biomechanicaltesting which confirmed increases in Young's modulus (Wollensak, G. andSpoerl, E., “Collagen crosslinking of human and porcine sclera,” J.Cataract Refract. Surg. 2004; 30:689-95; Kohlhaas, M., et al.,“Biomechanical evidence of the distribution of cross-links in corneastreated with riboflavin and ultraviolet A light,” J. Cataract Refract.Surg. 2006; 32:279-283). Such basic biochemical, biomechanical, andanimal studies were then followed by in vivo experiments aimed atdetermining the potential usefulness of this treatment in the livinghuman eye.

Several chemical cross-linking agents were tested previously by the UVRgroup in comparison studies with the UVR method and included glucose,ribose, glyceraldehyde, and glutaraldehyde. Of these, onlyglyceraldehyde and glutaraldehyde, (i.e. aldehydes) were found toproduce a significant biomechanical effect (Wollensak, G. and Spoerl,E., 2004). Glutaraldehyde is a well-known cross-linking agent used fortissue cross-linking of bioprosthetic heart valves and for tissuefixation prior to viewing by electron microscopy. Its utility as an invivo cross-linking agent, however, is limited by its significantcytotoxic effects. This is true for several other effective yet toxicaldehyde cross-linking agents, such as formaldehyde and glycoaldehyde.Glyceraldehyde is a physiologic metabolic product, is generallyconsidered non-toxic, and could also be potentially used for topicalcorneal cross-linking. Another class of cross-linking compounds thatcould have utility for in vivo cross-linking is the iridoid compounds,of which genipin is an example. Nimni, M. E., “Glutaraldehyde fixationrevisited,” Journal of Long-Term Effects of Medical Implants 2001;11(3&4):151-161; Jayakrishnan, A. and Jameela, S. R., “Review:Glutaraldehyde as a fixative in bioprostheses and drug deliverymatrices,” Biomaterials 1996; 17:471-484.

This invention uses a formaldehyde-donating compound (e.g., nitrogenoxide containing compounds), and in particular β-Nitro alcohols (βNAs)to cross-link collagen in collagenous tissue.

Nonenzymatic Cross-Linking of Collagen Tissue

There are two general types of collagen cross-linking: enzymatic andnon-enzymatic. Enzymatic cross-linking occurs in distinct telopeptideand helical regions of the collagen molecule. The process is mediated bythe enzyme lysyl oxidase and forms a number of borohydride reducible andnonreducible cross-links. Eyre, D. R., “Cross-linking in collagen andelastin.” Ann. Review Biochem. 53:717-48 (1984); Bailey, A. J.,“Glycation of collagen: the basis of its central role in the latecomplications of ageing and diabetes.” Mech. Ageing, Devel., 122:735-55(2001).

Nonenzymatic cross-linking is thought to accumulate gradually over thecourse of a person's lifetime, resulting in the hallmark aging change ofdecreased solubility and increased resistance to enzymatic digestion.This process is especially deleterious because the formation ofincreasingly undigestible collagen will prevent its clearance bycollagenases, further allowing for the accumulation of damaged proteins.Bailey, A. J., “Glycation of collagen: the basis of its central role inthe late complications of ageing and diabetes.” Mech. Ageing, Dovel.,122:735-55 (2001); Paul, R. G., and Bailey, “Glycation of collagen: thebasis of its central role in the late complications of ageing anddiabetes.” Int. J. Biochem. Cell Biol. 28(12):1297-1310 (1996).

Collagen cross-linking via nonenzymatic glycation (NEG) has beenimplicated in the development of increased tissue stiffness. Anadditional and novel mechanism that may contribute to nonenzymatic humancollagen cross-linking is through nonenzymatic nitrite (NEN)modification. Paik, D. C., et al., “The nitrite/collagen reaction:non-enzymatic nitration as a model system for age-related damage.” Con.Tis. Res. 42(2):111-22 (2001). NEN of type I collagen results in anincreased resistance to proteolytic digestion and alters theultraviolet/visible absorption consistent with aging changes.

Nitrite

Several methods are known to induce nonenzymatic cross-linking ofcollagenous tissues. The best known of these include methods involvingglutaraldehyde, sugar molecules such as glucose, ribose, glyceraldehyde,glycoaldehyde, rose bengal/white-light irradiation, and riboflavin withultraviolet-A irradiation. The latter method, riboflavin/UVA has beenused successfully in the treatment of keratoconus.

This invention is an alternative method of tissue cross-linking in theeye, that is, a reaction of collagen with the nitrite ion. Nitrite is acompound which has a significant history in the scientific literature.Nitrite, in the form of its sodium and/or potassium salt, has been usedfor several decades as a means of preserving and altering the qualitiesof meat and fish products in the process of curing. This processing iswell known to the meat industry and is performed specifically topreserve meats destined for human consumption. The process preventsspoilage by Clostridium botulinum, the bacterium causing the diseaseknown as botulism.

Nitrite reactions have been used to study the human aging process, sincenon-enzymatic collagen cross-linking is a hallmark change observedduring the aging of numerous organ systems, including the eye,cardiovascular system, and skin. Nitrite reactions with collagen andcollagenous engineered tissues result in the formation of non-enzymaticcross-linking and resultant tissue stiffening. Paik, D. C., et al.,“Nitrite-Induced Cross-Linking Alters Remodeling and MechanicalProperties of Collagenous Engineered Tissues.” Connective TissueResearch, 47:163-76 (2006). Although previous studies have viewednitrite mediated collagen cross-linking as a potential mechanism in thedevelopment of human disease, this invention provides potentialtherapeutic benefit that intentional nitriteinduced collagencross-linking may have. This concept has been spurred by recentdevelopments in the treatment of keratoconus. In this case, collagencross-linking using riboflavin/UVA has been used to stabilize cornealcollagen lamellae, preventing the untoward effects of progressivecorneal thinning. Thus, this invention involves the application ofnitrite induced cross-linking to the stiffening of collagen containingtissues for the purpose of stabilization with therapeutic intent.

The formation of reactive nitrogen species (RNS) and resultant nitrativeand nitrosative damage is important in this invention. Reactionsinvolving nitrite ion are a means through which nitrogen oxide exerteffects on human tissues. Nitrite can act as a reactive substrate for anarray of chemical reactions. At least three different reaction pathwaysare known to occur and have biological significance. First, nitrite canmediate nitrosation reactions under acidic conditions through theformation of nitrosonium ion (NO⁺) and/or dinitrogen trioxide (N₂O₃) inacidic electrophilic additions. Kurosky, A., and Hofmann, T. “Kineticsof the reaction of nitrous acid with model compounds and proteins, andthe conformational state of N-terminal groups in the chymotrypsinfamily.” Can. J. Biochem. 50:1282-96 (1972); Zhang, Y. Y., et al.,“Nitrosation of tryptophan residue(s) in serum albumin and modeldipeptides.” J. Biol. Chem. 271(24):14271-79. Second, oxometal complexformation by heme peroxidase/H₂O₂, or free Fe/H₂O₂ in Fenton-typereactions, can cause nitration through single electron transfer tonitrite, forming nitrogen dioxide (NO₂). Finally, hydroxyl radical (OH)and nitrogen dioxide (NO₂) can be produced through the photochemicaldecomposition of nitrite. Thus, nitrite can be involved in bothnitrosation as well as nitration reactions.

Nitrites of the alkali and alkaline earth metals may be synthesized byreacting a mixture of nitric oxide and nitrogen dioxide with thecorresponding metal hydroxide solution, as well as through thedecomposition of the corresponding nitrate. Nitrites are also availablethrough the reduction of the corresponding nitrates. Nitrites can beobtained from sources such as nitrous acid (as nitrosonium ion and/ordinitrogen trioxide); nitrosyl halides of the formula HalNO where Hal isa member of the group of fluorine, chlorine, bromine, iodine, orastatine; nitrosonium salts; alkyl nitrites; N-Nitrososulfonamides ofthe formula RSO₂N(NO)R′, where R and R′ can be any organic substituent,such as an acetal, acid anhydride, alcohol, aldehyde, alkane,cycloalkane, alkene, cycloalkene, alkyl, alkylamine, alkyl halide,alkyne, cycloalkyne, allyl, amide, amine, annulene, arene, aryl halide,arylamine, aryne, carbinolamine, carboxylic acid, dicarboxilic acid,ether, hydrocarbon, imide, imine, ketone, lactam, lactone, peroxide,phenol, phenyl, polyamide, polyamine, polycyclic aromatic hydrocarbon,polycyclic hydrocarbon, saccharide, thiol, or thioester;tetranitromethane C(NO₂)₄; inorganic nitrates; nitrite with carbonylgroup catalysts; nitrosyl carboxylates (acyl nitrites) of the formulaRCOONO, where R has the same definition as previously disclosed; Fremy'ssalt K₂[(SO₃)₂NO]; and sulfur-nitroso compounds such as thionylchloronitrite SOCIONO and thionyl dinitrite SO(ONO)₂.

Nitrite ion is also one of the many by-products of nitric oxide, and isknown to accumulate in the anterior chamber of the eye during ocularinflammation and can be derived from cigarette smoking. It has beenobserved that nitrite reactions with the matrix proteins elastin andcollagen produce damaging effects that mimic those observed in age- andsmoking-related illnesses. Since human exposure to nitrite is increasedby cigarette smoking, this reaction could explain the associationbetween ocular degeneration and smoking. Paik, D. C. and Dillon, J.,“The nitrite/alpha cristillin reaction: A possible mechanism in lensmatrix damage.” Exp. Eye Res., 70:73-80 (2000).

However, in some cases collagen cross-linking is desirable as atreatment of certain conditions or to preserve tissue duringtransplantation as described herein.

Formaldehyde-donating compounds (e.g., nitrogen oxide-containingcompounds) such as βNAs may be used as a means to facilitatecross-linking collagenous tissue under physiological pH and temperature,thereby making them suitable candidates for in vivo (e.g., ophthalmic)administration. Furthermore, formaldehyde-donating compounds (e.g.,nitro alcohols such as βNAs) generally demonstrate low toxicity and aretherefore amenable to clinical use (e.g., in vivo ophthalmicadministration).

The present inventors have disclosed the underlying mechanisms by whichformaldehyde-donating compounds (e.g., nitrogen oxide-containingcompounds such as βNAs) crosslink collagenous tissues, as well as meansfor catalyzing the catalytic effects of such nitrogen oxide-containingcompounds. Specifically, the instant studies have demonstrated thatformaldehyde is directly produced from certain formaldehyde-donatingcompounds (e.g., βNAs) by the reverse Henry reaction.Formaldehyde-donating compounds such as βNAs therefore act as aformaldehyde donor, capable of cross-linking collagenous tissues underphysiological conditions.

Also disclosed herein are compositions and methods for catalyzing thecross-linking of collagenous tissues. For example, the administration ofnitrogen oxide-containing compounds in the presences of one or morecatalysts (e.g., bases, DNA and/or enzymes) may be used as a means ofcatalyzing the cross-linking of collagenous tissues. Contemplatedcatalysts capable of driving the reverse Henry reaction include exposureto basic or alkaline conditions (e.g., exposure to NaHCO₃), DNA (e.g.,salmon testes DNA) and/or enzymes (e.g., hydroxynitrile lyases,transglutaminases and hydrolases). The present findings thereforeindicate that the formaldehyde-donating compounds (e.g., nitrogenoxide-containing compounds such as nitro alcohols or βNAs) representpromising, self-administered cross-linking clinical candidates suitablefor the in vivo induction of cross-linking of collagenous tissues (e.g.,tissue in the cornea).

Additional references relating to this invention include the following:Abraham, V. C., et al., “High content screening applied to large-scalecell biology,” Trends in Biotechnology 2004; 22(1):15-22; Amano, S., etal., “Comparison of central corneal thickness measurements by rotatingscheimpflug camera, ultrasonic pachymetry, and scanning-slit cornealtopography,” Ophthalmology 2006; 113:937-941; Bailey, A. J., “Molecularmechanisms of ageing in connective tissues,” Mech. Aging Dev. 2001;122:735-55; Banse, X., et al., “Cross-link profile of bone collagencorrelates with structural organization of trabeculae,” Bone 2002;31(1):70-76; Bednarz, J., et al., “Effect of three different media onserum free culture of donor corneas and isolated human cornealendothelial cells,” Br. J. Ophthalmol. 2001; 85:1416-1420; Brady, J. D.and Robins, S. P., “Structural characterization of pyrrolic cross-linksin collagen using a biotinylated Ehrlich's reagent,” J. Biol. Chem.2001; 276(22):18812-18818; Chiou, A. G. Y., et al., “Clinical cornealconfocal microscopy,” Survey of Ophthalmology 2006; 51(5):482-500; Eyre,D. R., et al., “Cross-linking in collagen and elastin,” Ann. Rev.Biochem. 1984; 53:717-748; Lackner, B., et al., “Repeatability andreproducibility of central corneal thickness measurement with pentacam,orbscan, and ultrasound,” Optometry and Vision Science 2005; 82:892-899;Lee, M. Y. and Dordick, J. S., “High-throughput human metabolism andtoxicity analysis,” Current Opinion in Biotechnology 2006; 17:619-627;McLaren, J. W., et al., “Corneal thickness measurement of confocalmicroscopy, ultrasound, and scanning slit methods,” Am. J. Ophthalmol.2004; 137:1011-1020; Naor, J., et al., “Corneal endothelial cytotoxicityof diluted providone-iodine,” J. Cataract Refract. Surg. 2001;27:941-947; Sady, C., et al., “Advanced Maillard reaction andcrosslinking of corneal collagen in diabetes,” Biochem. Biophys. Res.Corn. 1995; 214(3):793-797; Sell, D. R. and Monnier, V. M., “Structureelucidation of a senescence cross-link from human extracellular matrix,”J. Biol. Chem. 1989; 264(36):21597-21602; Skinner, S. J. M., “Rapidmethod for the purification of the elastin cross-links, desmosine andisodesmosine,” J. Chromatog. 1982; 229:200-204; Wollensak, G., et al.,“Stress-strain measurements of human and porcine corneas afterriboflavin-ultraviolet-A-induced cross-linking,” J. Cataract Refract.Surg. 2003; 29:1780-1785.

What is claimed is:
 1. A method of accelerating the rate at which one or more formaldehyde-donating compounds cross-link collagen in a collagenous tissue comprising a step of contacting the one or more formaldehyde-donating compounds with one or more catalysts; wherein the one or more catalysts accelerate the rate at which formaldehyde is released from the one or more formaldehyde-donating compounds; wherein the accelerated release of formaldehyde from the one or more formaldehyde-donating compounds accelerates the rate at which collagen is cross-linked in the collagenous tissue; and wherein the catalyst is a base selected from the group consisting of aqueous solutions of NaHCO₃, KOH, NaOH, Ca(OH)₂, Na₂CO₃, KF, Bu₃P, MeONa/MeOH, and Et₃N, an enzyme selected from the group consisting of hydroxynitrile lyases, transglutaminases, and hydrolases, a double-stranded DNA of salmon sperm, a metal-based chiral catalyst, an organocatalyst, or a tetraaminophosphonium salt.
 2. The method of claim 1, wherein the formaldehyde-donating compound comprises a β-nitro alcohol and wherein the β-nitro alcohol is 2-nitroethanol.
 3. The method of claim 1, wherein the formaldehyde-donating compound comprises a β-nitro alcohol and wherein the β-nitro alcohol is 2-nitro-1-propanol.
 4. The method of claim 1, wherein the formaldehyde-donating compound comprises a β-nitro alcohol and wherein the β-nitro alcohol is 2-hydroxymethyl-2-nitro-1,3-propanediol.
 5. The method of claim 1 wherein the formaldehyde-donating compound comprises a β-nitro alcohol and wherein the β-nitro alcohol is 2-methyl-2-nitro-1,3-propanediol.
 6. The method of claim 1 wherein the formaldehyde-donating compound comprises a β-nitro alcohol and wherein the β-nitro alcohol is 2-bromo-2-nitro-1,3-propanediol.
 7. The method of claim 1, wherein the collagenous tissue is cornea.
 8. A method of decelerating the rate at which one or more β-nitro alcohols cross-link collagen in a collagenous tissue comprising a step of contacting the one or more β-nitro alcohols with one or more enzymes; wherein the one or more enzymes decelerate the rate at which formaldehyde is released from the one or more β-nitro alcohols; wherein the decelerated release of formaldehyde from the one or more β-nitro alcohols decelerates the rate at which collagen is cross-linked in the collagenous tissue; and wherein the enzyme is selected from the group consisting of hydroxynitrile lyases, transglutaminases, and hydrolases.
 9. A method of treating a condition associated with a loss of structural integrity of a collagenous tissue, the method comprising a step of contacting the collagenous tissue with a composition; wherein the composition comprises (a) one or more β-nitro alcohols, and (b) one or more catalysts; wherein the composition improves the loss of structural integrity of the collagenous tissue by cross-linking the collagen in the collagenous tissue; and wherein the catalyst is a base selected from the group consisting of aqueous solutions of NaHCO₃, KOH, NaOH, Ca(OH)₂, Na₂CO₃, KF, Bu₃P, MeONa/MeOH, and Et₃N, an enzyme selected from the group consisting of hydroxynitrile lyases, transglutaminases, and hydrolases, a double-stranded DNA of salmon sperm, a metal-based chiral catalyst, an organocatalyst, or a tetraaminophosphonium salt.
 10. The method of claim 9, wherein the collagenous tissue is cornea.
 11. The method of claim 9, wherein the collagenous tissue is cornea and the condition associated with a loss of structural integrity of a collagenous tissue is selected from the group consisting of keratoconus, keratectasia and myopia.
 12. The method of claim 9, wherein the P-nitro alcohol is selected from the group consisting of 2-hydroxymethyl-2-nitro-1,3-propanediol, 2-methyl-2-nitro-1,3-propanediol, 2-bromo-2-nitro-1,3-propanediol, 2-nitroethanol, 2-nitro-1-propanol, 3-nitro-2-pentanol and 2-nitro-1-pentanol.
 13. The method of claim 9, wherein the catalyst is a base selected from the group consisting of aqueous solutions of NaHCO₃, KOH, NaOH, Ca(OH)₂, Na₂CO₃, KF, Bu₃P, MeONa/MeOH, and Et₃N, and the base accelerates the cross-linking of collagen in the collagenous tissue.
 14. The method of claim 13, wherein the base accelerates the cross-linking of collagen in the collagenous tissue by at least about two-fold.
 15. The method of claim 9, wherein the catalyst is a double-stranded DNA of salmon sperm, and the DNA accelerates the rate at which the one or more β-nitro alcohols cross-link collagen in a collagenous tissue.
 16. The method of claim 15, wherein the DNA accelerates the rate at which the one or more β-nitro alcohols cross-link collagen in a collagenous tissue by at least about two-fold.
 17. The method of 9, wherein the composition is selected from the group consisting of a solution, a suspension, an ointment, a gel or a cream.
 18. The method of claim 17, wherein the pH of the composition is alkaline.
 19. A method of cross-linking collagen in a collagenous tissue comprising contacting the collagenous tissue with a composition comprising (a) one or more β-nitro alcohols, and (b) one or more catalysts; wherein the composition effectuates the cross-linking of the collagen in the collagenous tissue; and wherein the catalyst is a base selected from the group consisting of aqueous solutions of NaHCO₃, KOH, NaOH, Ca(OH)₂, Na₂CO₃, KF, Bu₃P, MeONa/MeOH, and Et₃N, an enzyme selected from the group consisting of hydroxynitrile lyases, transglutaminases, and hydrolases, a double-stranded DNA of salmon sperm, a metal-based chiral catalyst, an organocatalyst, or a tetraaminophosphonium salt.
 20. The method of claim 19, wherein the collagenous tissue is in a subject.
 21. The method of claim 19, wherein the collagenous tissue is cornea.
 22. The method of claim 20, wherein the collagenous tissue is cornea and the subject is afflicted with one or more conditions selected from the group consisting of keratoconus, keratectasia and myopia.
 23. The method of claim 19, wherein the β-nitro alcohol is selected from the group consisting of 2-hydroxymethyl-2-nitro-1,3-propanediol, 2-methyl-2-nitro-1,3-propanediol, 2-bromo-2-nitro-1,3-propanediol, 2-nitroethanol, 2-nitro-1-propanol, 3-nitro-2-pentanol and 2-nitro-1-pentanol. 